Reversir tm compounds

ABSTRACT

The present invention relates, in general to agents that modulate the pharmacological activity of conjugated siRNAs.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 62/093,906, filed Dec. 18, 2014, and U.S.Provisional Application No. 62/238,467, filed Oct. 7, 2015, the contentsof both which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present disclosure relates generally to oligomeric compounds(oligomers), which target siRNAs (e.g. conjugated or unconjugatedsiRNAs) in vivo, thereby providing a method of tailored control of RNAipharmacology and therefore the therapeutic activity and/or side effectsof siRNA based therapeutics in vivo.

BACKGROUND

Conjugated and unconjugated siRNA compounds have been used to modulatetarget nucleic acids. Conjugated and unconjugated siRNAs comprising avariety of modifications and motifs have been reported. In certaininstances, such compounds are useful as research tools and astherapeutic agents.

SUMMARY OF THE INVENTION

In certain embodiments, provided herein are REVERSIR compounds (REVERSIRis a trademark of Alnylam Pharmaceuticals, Inc.). Such compounds reduceRNAi activity of a siRNA compound, for example conjugated siRNA orunconjugated siRNA. Generally, the REVERSIR compounds modulate hybridizeor bind siRNA molecule in a sequence dependent manner and modulate(e.g., inhibit or reverse) their activity.

In certain embodiments, the present invention provides REVERSIRcompounds that are complementary to at least one strand of siRNAcompounds (e.g. conjugated or unconjugated siRNA). In some embodiments,the REVERSIR compounds are complementary to the antisense strand ofsiRNA compounds. In some other embodiments, the REVERSIR compounds arecomplementary to the sense strand of siRNA compounds.

In certain embodiments, the present invention provides REVERSIRcompounds comprising a modified oligonucleotide consisting of 6 to 30linked nucleosides and having a nucleobase sequence substantiallycomplementary to at least one strand of siRNA compounds (e.g. conjugatedor unconjugated siRNA). In some embodiments, the REVERSIR compoundscomprise a modified oligonucleotide consisting of 6 to 30 linkednucleosides and having a nucleobase sequence substantially complementaryto the antisense strand of siRNA compounds. In some other embodiments,the REVERSIR compounds comprise a modified oligonucleotide consisting of6 to 30 linked nucleosides and having a nucleobase sequencesubstantially complementary to the sense strand of siRNA compounds.

In certain such embodiments, the modified oligonucleotide is asingle-stranded oligonucleotide and/or is at least 90% complementary toat least one strand of the siRNA. In some embodiments, the modifiedoligonucleotide is a single-stranded oligonucleotide and/or is at least90% complementary to the antisense strand of the siRNA. In some otherembodiments, the modified oligonucleotide is a single-strandedoligonucleotide and/or is at least 90% complementary to the sense strandof the siRNA.

In certain embodiments, the REVERSIR compound is fully complementary toat least one strand of the conjugated or unconjugated siRNA. In someembodiments, the REVERSIR compound is fully complementary to theantisense strand of the siRNA. In some other embodiments, the REVERSIRcompound is fully complementary to the sense strand of the siRNA.

In certain embodiments, REVERSIR compounds comprise at least onemodified internucleoside or intersugar linkage. In certain suchembodiments, at least one (e.g., one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen ormore and upto and including all) internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, REVERSIR compounds comprise at least onenucleoside comprising a modified sugar. In certain such embodiments, themodified sugar is a bicyclic sugar or sugar comprising a 2′-O-methyl ora 2′-O-methoxyethyl.

In certain embodiments, REVERSIR compounds comprise one or more (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen or more and upto and including all)locked nucleic acid (LNA) monomers.

In certain embodiments, REVERSIR compounds comprise at least one (e.g.,one, two, three, four, five, six, seven, eight, nine, ten or more)nucleotide that does not comprise a 2′-O-methyl group, i.e., theREVERSIR compound is not fully 2′-O-methyl. In some embodiments, eachnucleoside in the REVERSIR compound is a 2′-O-methyl nucleoside and theREVERSIR compound comprises at least one (e.g., one, two, three, four,five, six, seven, eight, nine, ten or more) G-clamp nucleobases.

In certain embodiments, REVERSIR compounds comprise at least onenucleoside comprising a modified nucleobase. In certain suchembodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, REVERSIR compounds comprise at least onemodification. In certain such embodiments, REVERSIR compounds compriseone or more nucleoside modifications and or one or more linkagemodifications. In certain embodiments, REVERSIR compounds comprise oneor more modifications selected from: sugar modifications, linkagemodifications, nucleobase modifications, conjugates (e.g., ligands), andany combinations thereof.

In certain embodiments, REVERSIR compounds comprise a modifiedoligonucleotide comprising: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.

In certain embodiments, REVERSIR compounds comprise a modifiedoligonucleotide comprising: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; and wherein each internucleosidelinkage is a phosphorothioate linkage.

In certain embodiments, REVERSIR compound comprises a modifiedoligonucleotide consisting of 6-17, 7-16 8-15 or 6-25 linkednucleosides. In some embodiments, REVERSIR compound comprises a modifiedoligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15 or 20 linkednucleosides.

In certain embodiments, REVERSIR compound comprises a modifiedoligonucleotide wherein each nucleoside is modified.

In some embodiments, REVERSIR compound comprises or consists of ninelinked nucleosides.

In some embodiments, REVERSIR compound has low PS content. By low PScontent is meant that the REVERSIR compound has 1, 2, 3, 4 or 5phosphorothioate linkages per nine nucleosides of the REVERSIR compound.

In some embodiments, REVERSIR compound comprises or consists of ninelinked nucleosides and has low PS content.

In some embodiments, REVERSIR compound consists of nine linkednucleosides and comprises five phosphorothioate linkages.

In some embodiments, REVERSIR compound consists of nine linkednucleosides, comprises five phosphorothioate linkages and is linked to aligand.

In certain embodiments, REVERSIR compounds are complementary to theantisense or sense strand of a conjugated or unconjugated siRNA, whereinthe siRNA is targeted to an mRNA. In certain embodiments, the siRNA istargeted to an mRNA encoding a blood factor. In certain embodiments, thesiRNA is targeted to an mRNA encoding a protein involved in metabolism.In certain embodiments, the siRNA is targeted to an mRNA encoding aprotein involved in diabetes. In certain embodiments, the siRNA istargeted to an mRNA encoding a protein involved in cardiopathology. Incertain embodiments, the siRNA is targeted to an mRNA encoding a proteinexpressed in nerve cells. In certain embodiments, the siRNA is targetedto an mRNA encoding a protein expressed in the central nervous system.In certain embodiments, the siRNA is targeted to an mRNA expressed inperipheral nerves.

In certain embodiments, the conjugated or unconjugated siRNA is targetedto an mRNA encoding a protein expressed in the liver. In certainembodiments, the siRNA is targeted to an mRNA encoding a proteinexpressed in the kidney.

In certain embodiments, the conjugated or unconjugated siRNA is targetedto a pre-mRNA. In certain embodiments, the conjugated or unconjugatedsiRNA is targeted to a micro-RNA. In certain embodiments, the conjugatedor unconjugated siRNA activates the RISC pathway. In some embodiments,the conjugated or unconjugated siRNA inhibits the expression of a targetnucleic acid.

In certain embodiments, REVERSIR compounds modulate the RISC pathway. Insome embodiments, REVERSIR compounds inhibit the RISC pathway.

In certain embodiments, the invention provides a composition comprisinga REVERSIR compound or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier or diluent.

In certain embodiments, the invention provides methods comprisingadministering to a subject (e.g., an animal) a REVERSIR compound orcomposition comprising same. In certain embodiments, the subject is ahuman. In certain embodiments, the administering is oral, topical, orparenteral.

In certain embodiments, the invention provides methods of inhibitingRNAi activity of a conjugated or unconjugated siRNA in a cell. Themethod, generally, comprises contacting the cell with a REVERSIRcompound according the present invention and thereby inhibiting the RNAiactivity in the cell. In certain such embodiments, the cell is in invivo. In some embodiments, the cell is in vitro. In some embodiments thecell is ex vivo. In some embodiments, the cell is in a subject. In somefurther embodiments of this, the cell is an animal. In certainembodiments, the animal is a human.

In certain embodiments, the invention provides methods comprising:contacting a cell with a conjugated or unconjugated siRNA; detectingRNAi activity; and contacting the cell with a REVERSIR compound. Incertain embodiments, the method the detecting RANi activity comprisesmeasuring the amount of target mRNA present, the amount of targetprotein present, and/or the activity of a target protein. In certainembodiments, such methods comprise detecting REVERSIR activity bymeasuring RNAi activity after contacting the cell with the REVERSIRcompound. In certain such methods, the cell is in vivo. In someembodiments, the cell is in an animal. In certain embodiments, theanimal is a human.

In certain embodiments, the invention provides methods of ameliorating aside-effect of siRNA treatment comprising: contacting a cell with aconjugated or unconjugated siRNA; detecting a side-effect; contactingthe cell with a REVERSIR compound; and thereby ameliorating the sideeffect of the siRNA.

In certain embodiments, the invention provides methods of treating apatient comprising: administering to the patient a conjugated orunconjugated siRNA; monitoring the patient for siRNA activity; and ifthe siRNA activity becomes higher than desired, administrating aREVERSIR compound. In certain such embodiments, the monitoring siRNAactivity comprises measuring the amount of target mRNA present,measuring the amount of target protein present and/or measuring theactivity of a target protein. In certain embodiments, such methodsinclude detecting REVERSIR activity by measuring siRNA activity afteradministration of the REVERSIR compound. In certain embodiments, thepatient is a mammal. In some embodiments, the patient is a human.

In certain embodiments, the invention provides methods of treating apatient comprising: administering to the patient a conjugated orunconjugated siRNA; monitoring the patient for one or more side effect;and if the one or more side effect reaches an undesirable level,administrating a REVERSIR compound. In certain embodiments, the patientis a mammal. In some embodiments, the patient is a human.

In certain embodiments, the invention provides a kit comprising aconjugated or unconjugated siRNA and a REVERSIR compound; REVERSIRcompound and a non-oligomeric REVERSIR; or conjugated or unconjugatedsiRNA compound, REVERSIR compound, and a non-oligomeric REVERSIR. Incertain such embodiments, the non-oligomeric REVERSIR is a targetprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in vivo activity of exemplary REVERSIR compounds targetingantithrombin (AT) siRNAs.

FIG. 2 shows that reversal of activity of siRNAs by REVERSIR compoundsin vivo is rapid and dose-dependent. Full reversal can be seen within4-days of dosing.

FIG. 3 shows the effect of REVERSIR compound length on the in vivoactivity of exemplary REVERSIR compounds. As seen, shorter REVERSIRcompounds showed better in vivo activity than the longer REVERSIRcompounds.

FIG. 4 shows the effect of exemplary nucleic acid modifications on thein vivo activity of REVERSIR compounds.

FIG. 5 shows the effect of number of phosphorothioate internucleosidelinkages on the in vivo activity of REVERSIR compounds.

FIGS. 6 and 7 show that REVERSIR compounds have increased in vivopotency with decreasing length

FIGS. 8 and 9 shows effect of number phosphorothioate linkages on theactivity of REVERSIR compounds.

FIG. 10 shows further improvement in potency for exemplary REVERSIRcompounds.

FIG. 11 shows in vitro reversal of siRNA activity by free uptake ofexemplary REVERSIR compounds targeting antithrombin siRNA in primarymouse hepatocytes.

FIG. 12 shows in vitro reversal of siRNA activity by free uptake ofexemplary REVERSIR compounds targeting antithrombin siRNA in primarymouse hepatocytes at various concentrations.

FIGS. 13 and 14 show in vitro reversal of siRNA activity by free uptakeof exemplary REVERSIR compounds targeting Factor IX siRNAs in primarymouse hepatocytes at various concentrations.

FIG. 15 shows the effect of high-affinity chemistry on the in vivoactivity of exemplary REVERSIR compounds targeting Factor IX siRNAs.

FIGS. 16 and 17 show the effect of REVERSIR compound length on the invivo activity of exemplary REVERSIR compounds targeting Factor IXsiRNAs. REVERSIR compounds were administered at 3 mg/kg (FIG. 16) and 1mg/kg (FIG. 17)

FIG. 18 shows the effect of linker, between the REVERSIR compound andthe ligand conjugated with the REVERSIR compound, on the in vivoactivity of exemplary REVERSIR compounds targeting Factor IX siRNAs.

FIG. 19 shows the effect of phosphorothioate linkages in the REVERSIRcompound on the in vivo activity of exemplary REVERSIR compoundstargeting Factor IX siRNAs.

FIG. 20 shows the effect of linker, between the REVERSIR compound andthe ligand conjugated with the REVERSIR compound, on the in vitroactivity of exemplary REVERSIR compounds targeting Factor IX siRNAs.

FIG. 21 shows the effect on activity of siRNA by exemplary REVERSIRcompounds matching certain portion of the antisense strand of the siRNA.

FIG. 22 shows in vivo dos-dependent effect of exemplary REVERSIRcompounds targeting Factor IX siRNA.

FIG. 23 shows that REVERSIR compounds are tolerated in vivo.

FIGS. 24A and 24B show in vivo reversal of siRNA activity by someexemplary REVERSIR compounds in non-human primates.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,18th edition, 1990; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosureherein are incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, the term “nucleoside” means a glycosylamine comprising anucleobase and a sugar. Nucleosides includes, but are not limited to,naturally occurring nucleosides, abasic nucleosides, modifiednucleosides, and nucleosides having mimetic bases and/or sugar groups.

As used herein, the term “nucleotide” refers to a glycosomine comprisinga nucleobase and a sugar having a phosphate group covalently linked tothe sugar. Nucleotides may be modified with any of a variety ofsubstituents.

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. A nucleobase may comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “heterocyclic base moiety” refers to anucleobase comprising a heterocycle.

As used herein, the term “oligomeric compound” refers to a polymericstructure comprising two or more sub-structures and capable ofhybridizing to a region of a nucleic acid molecule. In certainembodiments, oligomeric compounds are oligonucleosides. In certainembodiments, oligomeric compounds are oligonucleotides. In certainembodiments, oligomeric compounds are antisense compounds. In certainembodiments, oligomeric compounds are REVERSIR compounds. In certainembodiments, oligomeric compounds comprise conjugate groups.

As used herein “oligonucleoside” refers to an oligonucleotide in whichthe internucleoside linkages do not contain a phosphorus atom.

As used herein, the term “oligonucleotide” refers to an oligomericcompound comprising a plurality of linked nucleosides. In certainembodiment, one or more nucleotides of an oligonucleotide is modified.In certain embodiments, an oligonucleotide comprises ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). In certain embodiments,oligonucleotides are composed of naturally- and/ornon-naturally-occurring nucleobases, sugars and covalent internucleosidelinkages, and may further include non-nucleic acid conjugates.

As used herein “internucleoside linkage” refers to a covalent linkagebetween adjacent nucleosides.

As used herein “naturally occurring internucleoside linkage” refers to a3′ to 5′ phosphodiester linkage.

As used herein the term “detecting siRNA activity” or “measuring siRNAactivity” means that a test for detecting or measuring siRNA activity isperformed on a particular sample and compared to that of a controlsample. Such detection and/or measuring can include values of zero.Thus, if a test for detection of siRNA activity results in a finding ofno siRNA activity (siRNA activity of zero), the step of “detecting siRNAactivity” has nevertheless been performed.

As used herein the term “control sample” refers to a sample that has notbeen contacted with a reporter oligomeric compound.

As used herein, the term “motif” refers to the pattern of unmodified andmodified nucleotides in an oligomeric compound.

As used herein, the term “REVERSIR compound” refers to an oligomericcompound that is complementary to and capable of hybridizing with atleast one strand of a conjugated or unconjugated siRNA. Withoutlimitations, the REVERSIR compound could not only block unintendedtarget PD effect but also block any potential off-target activity thatcould happen with a conjugated or unconjugated siRNA.

As used herein, the term “non-oligomeric REVERSIR” refers to a compoundthat does not hybridize with a strand of siRNA and that reduces theamount or duration of a siRNA activity. In certain embodiments, anon-oligomeric REVERSIR is a target protein.

As used herein, the term “REVERSIR activity” refers to any decrease inintensity or duration of any siRNA activity attributable tohybridization of a REVERSIR compound to one of the strands of the siRNA.

As used herein, the term “chimeric oligomer” refers to an oligomericcompound, having at least one sugar, nucleobase or internucleosidelinkage that is differentially modified as compared to at least on othersugar, nucleobase or internucleoside linkage within the same oligomericcompound. The remainder of the sugars, nucleobases and internucleosidelinkages can be independently modified or unmodified, the same ordifferent.

As used herein, the term “chimeric oligonucleotide” refers to anoligonucleotide, having at least one sugar, nucleobase orinternucleoside linkage that is differentially modified as compared toat least on other sugar, nucleobase or internucleoside linkage withinthe same oligonucleotide. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodified,the same or different.

As used herein, the term “mixed-backbone oligomeric compound” refers toan oligomeric compound wherein at least one internucleoside linkage ofthe oligomeric compound is different from at least one otherinternucleoside linkage of the oligomeric compound.

As used herein, the term “target protein” refers to a protein, themodulation of which is desired.

As used herein, the term “target gene” refers to a gene encoding atarget protein.

As used herein, the term “target nucleic acid” refers to any nucleicacid molecule the expression or activity of which is capable of beingmodulated by a conjugated or unconjugated siRNA compound. Target nucleicacids include, but are not limited to, RNA (including, but not limitedto pre-mRNA and mRNA or portions thereof) transcribed from DNA encodinga target protein, and also cDNA derived from such RNA, and miRNA. Forexample, the target nucleic acid can be a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent.

As used herein, the term “target siRNA” refers to a siRNA compound thatis targeted by a REVERSIR compound.

As used herein, the term “targeting” or “targeted to” refers to theassociation of antisense strand of a siRNA to a particular targetnucleic acid molecule or a particular region of nucleotides within atarget nucleic acid molecule.

As used herein, the term “nucleobase complementarity” refers to anucleobase that is capable of base pairing with another nucleobase. Forexample, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.

As used herein, the term “non-complementary nucleobase” refers to a pairof nucleobases that do not form hydrogen bonds with one another orotherwise support hybridization.

As used herein, the term “complementary” refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an oligomeric compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are oligomeric compounds (e.g.,REVERSIR compounds, siRNAs, and the like) that may comprise up to about20% nucleotides that are mismatched (i.e., are not nucleobasecomplementary to the corresponding nucleotides of the target).Preferably the oligomeric compounds, such as REVERSIR compounds andsiRNAs, contain no more than about 15%, more preferably not more thanabout 10%, most preferably not more than 5% or no mismatches. Theremaining nucleotides are nucleobase complementary or otherwise do notdisrupt hybridization (e.g., universal bases). One of ordinary skill inthe art would recognize the compounds provided herein are at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% complementary to a target nucleic acid.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense strand of a siRNA and itstarget nucleic acid or a REVERSIR to its target siRNA). While notlimited to a particular mechanism, the most common mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleobases). For example, the natural base adenine isnucleobase complementary to the natural nucleobases thymidine and uracilwhich pair through the formation of hydrogen bonds. The natural baseguanine is nucleobase complementary to the natural bases cytosine and5-methyl cytosine. Hybridization can occur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the abilityof an oligomeric compound to hybridize to one nucleic acid site withgreater affinity than it hybridizes to another nucleic acid site. Incertain embodiments, the antisense strand of an siRNA specificallyhybridizes to more than one target site.

As used herein, “designing” or “designed to” refer to the process ofdesigning an oligomeric compound that specifically hybridizes with aselected nucleic acid molecule.

As used herein, the term “modulation” refers to a perturbation offunction or activity when compared to the level of the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing.

As used herein, the term “expression” refers to all the functions andsteps by which a gene's coded information is converted into structurespresent and operating in a cell. Such structures include, but are notlimited to the products of transcription and translation.

As used herein, “variant” refers to an alternative RNA transcript thatcan be produced from the same genomic region of DNA. Variants include,but are not limited to “pre-mRNA variants” which are transcriptsproduced from the same genomic DNA that differ from other transcriptsproduced from the same genomic DNA in either their start or stopposition and contain both intronic and exonic sequence. Variants alsoinclude, but are not limited to, those with alternate splice junctions,or alternate initiation and termination codons.

As used herein, “high-affinity modified monomer” refers to a monomerhaving at least one modified nucleobase, internucleoside linkage orsugar moiety, when compared to naturally occurring monomers, such thatthe modification increases the affinity of an antisense compoundcomprising the high-affinity modified monomer to its target nucleicacid. High-affinity modifications include, but are not limited to,monomers (e.g., nucleosides and nucleotides) comprising 2′-modifiedsugars.

As used herein, the term “2′-modified” or “2′-substituted” means a sugarcomprising substituent at the 2′ position other than H or OH.2′-modified monomers, include, but are not limited to, BNA's andmonomers (e.g., nucleosides and nucleotides) with 2′-substituents, suchas allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF3,O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(Rm)(Rn), or O—CH₂—C(═O)N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted orunsubstituted C₁-C₁₀ alkyl. In certain embodiments, oligomeric compoundscomprise a 2′ modified monomer that does not have the formula2′-O(CH₂)_(n)H, wherein n is one to six. In certain embodiments,oligomeric compounds comprise a 2′ modified monomer that does not havethe formula 2′-OCH₃. In certain embodiments, oligomeric compoundscomprise a 2′ modified monomer that does not have the formula or, in thealternative, 2′-O(CH₂)₂OCH₃.

As used herein, the term “locked nucleic acid” or “LNA” or “lockednucleoside” or “locked nucleotide” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside includes a bridgeconnecting two carbon atoms on the furanose ring, thereby forming abicyclic ring system. Locked nucleic acids are also referred to asbicyclic nucleic acids (BNA).

As used herein, unless otherwise indicated, the term “methyleneoxy LNA”alone refers to β-D-methyleneoxy LNA.

As used herein, the term “MOE” refers to a 2′-O-methoxyethylsubstituent.

As used herein, the term “gapmer” refers to a chimeric oligomericcompound comprising a central region (a “gap”) and a region on eitherside of the central region (the “wings”), wherein the gap comprises atleast one modification that is different from that of each wing. Suchmodifications include nucleobase, monomeric linkage, and sugarmodifications as well as the absence of modification (unmodified). Thus,in certain embodiments, the nucleotide linkages in each of the wings aredifferent than the nucleotide linkages in the gap. In certainembodiments, each wing comprises nucleotides with high affinitymodifications and the gap comprises nucleotides that do not comprisethat modification. In certain embodiments the nucleotides in the gap andthe nucleotides in the wings all comprise high affinity modifications,but the high affinity modifications in the gap are different than thehigh affinity modifications in the wings. In certain embodiments, themodifications in the wings are the same as one another. In certainembodiments, the modifications in the wings are different from eachother. In certain embodiments, nucleotides in the gap are unmodified andnucleotides in the wings are modified. In certain embodiments, themodification(s) in each wing are the same. In certain embodiments, themodification(s) in one wing are different from the modification(s) inthe other wing. In certain embodiments, oligomeric compounds are gapmershaving 2′-deoxynucleotides in the gap and nucleotides with high-affinitymodifications in the wing.

As used herein, the term “prodrug” refers to a therapeutic agent that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts of active compounds that retain the desired biological activity ofthe active compound and do not impart undesired toxicological effectsthereto.

As used herein, the term “cap structure” or “terminal cap moiety” refersto chemical modifications, which have been incorporated at eitherterminus of an antisense compound.

As used herein, the term “prevention” refers to delaying or forestallingthe onset or development of a condition or disease for a period of timefrom hours to days, preferably weeks to months.

As used herein, the term “amelioration” refers to a lessening of atleast one activity or one indicator of the severity of a condition ordisease. The severity of indicators may be determined by subjective orobjective measures which are known to those skilled in the art.

As used herein, the term “treatment” refers to administering acomposition of the invention to effect an alteration or improvement ofthe disease or condition. Prevention, amelioration, and/or treatment mayrequire administration of multiple doses at regular intervals, or priorto onset of the disease or condition to alter the course of the diseaseor condition. Moreover, a single agent may be used in a singleindividual for each prevention, amelioration, and treatment of acondition or disease sequentially, or concurrently.

As used herein, the term “pharmaceutical agent” refers to a substancethat provides a therapeutic benefit when administered to a subject. Incertain embodiments, a pharmaceutical agent is an active pharmaceuticalagent. In certain embodiments, a pharmaceutical agent is a prodrug.

As used herein, the term “therapeutically effective amount” refers to anamount of a pharmaceutical agent that provides a therapeutic benefit toan animal.

As used herein, “administering” means providing a pharmaceutical agentto an animal, and includes, but is not limited to administering by amedical professional and self-administering.

As used herein, the term “co-administering” means providing more thanone pharmaceutical agent to an animal. In certain embodiments, such morethan one pharmaceutical agents are administered together. In certainembodiments, such more than one pharmaceutical agents are administeredseparately. In certain embodiments, such more than one pharmaceuticalagents are administered at the same time. In certain embodiments, suchmore than one pharmaceutical agents are administered at different times.In certain embodiments, such more than one pharmaceutical agents areadministered through the same route of administration. In certainembodiments, such more than one pharmaceutical agents are administeredthrough different routes of administration. In certain embodiments, suchmore than one pharmaceutical agents are contained in the samepharmaceutical formulation. In certain embodiments, such more than onepharmaceutical agents are in separate formulations.

As used herein, the term “pharmaceutical composition” refers to amixture of substances suitable for administering to an individual. Forexample, a pharmaceutical composition may comprise an antisenseoligonucleotide and a sterile aqueous solution. In certain embodiments,a pharmaceutical composition includes a pharmaceutical agent and adiluent and/or carrier.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within an organism (e.g. animal or a plant).As used herein, the term “ex vivo” refers to cells which are removedfrom a living organism and cultured outside the organism (e.g., in atest tube). As used herein, the term “in vivo” refers to events thatoccur within an organism (e.g. animal, plant, and/or microbe).

As used herein, the term “subject” or “patient” refers to any organismto which a composition disclosed herein can be administered, e.g., forexperimental, diagnostic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans) and/or plants. Usually the animal is a vertebratesuch as a primate, rodent, domestic animal or game animal. Primatesinclude chimpanzees, cynomologous monkeys, spider monkeys, and macaques,e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbitsand hamsters. Domestic and game animals include cows, horses, pigs,deer, bison, buffalo, feline species, e.g., domestic cat, caninespecies, e.g., dog, fox, wolf, avian species, e.g., chicken, emu,ostrich, and fish, e.g., trout, catfish and salmon. Patient or subjectincludes any subset of the foregoing, e.g., all of the above, butexcluding one or more groups or species such as humans, primates orrodents. In certain embodiments of the aspects described herein, thesubject is a mammal, e.g., a primate, e.g., a human. The terms,“patient” and “subject” are used interchangeably herein. A subject canbe male or female.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of humandiseases and disorders. In addition, compounds, compositions and methodsdescribed herein can be used to with domesticated animals and/or pets.

In one embodiment, the subject is human. In another embodiment, thesubject is an experimental animal or animal substitute as a diseasemodel. The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be covered. Examples of subjects include humans, dogs, cats,cows, goats, and mice. The term subject is further intended to includetransgenic species. In some embodiments, the subject can be of Europeanancestry. In some embodiments, the subject can be of African Americanancestry. In some embodiments, the subject can be of Asian ancestry.

In jurisdictions that forbid the patenting of methods that are practicedon the human body, the meaning of “administering” of a composition to ahuman subject shall be restricted to prescribing a controlled substancethat a human subject will self-administer by any technique (e.g.,orally, inhalation, topical application, injection, insertion, etc.).The broadest reasonable interpretation that is consistent with laws orregulations defining patentable subject matter is intended. Injurisdictions that do not forbid the patenting of methods that arepracticed on the human body, the “administering” of compositionsincludes both methods practiced on the human body and also the foregoingactivities.

As used herein, the term “parenteral administration,” refers toadministration through injection or infusion. Parenteral administrationincludes, but is not limited to, subcutaneous administration,intravenous administration, or intramuscular administration.

As used herein, the term “subcutaneous administration” refers toadministration just below the skin. “Intravenous administration” meansadministration into a vein.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

As used herein, the term “dosage unit” refers to a form in which apharmaceutical agent is provided. In certain embodiments, a dosage unitis a vial comprising lyophilized antisense oligonucleotide. In certainembodiments, a dosage unit is a vial comprising reconstituted antisenseoligonucleotide.

As used herein, the term “active pharmaceutical ingredient” refers tothe substance in a pharmaceutical composition that provides a desiredeffect.

As used herein, the term “side effects” refers to physiologicalresponses attributable to a treatment other than desired effects. Incertain embodiments, side effects include, without limitation, injectionsite reactions, liver function test abnormalities, renal functionabnormalities, liver toxicity, renal toxicity, central nervous systemabnormalities, and myopathies. For example, increased aminotransferaselevels in serum may indicate liver toxicity or liver functionabnormality. For example, increased bilirubin may indicate livertoxicity or liver function abnormality.

As used herein, the term “alkyl,” as used herein, refers to a saturatedstraight or branched hydrocarbon radical containing up to twenty fourcarbon atoms. Examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyland the like. Alkyl groups typically include from 1 to about 24 carbonatoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl)with from 1 to about 6 carbon atoms being more preferred. The term“lower alkyl” as used herein includes from 1 to about 6 carbon atoms.Alkyl groups as used herein may optionally include one or more furthersubstituent groups.

As used herein, the term “alkenyl,” as used herein, refers to a straightor branched hydrocarbon chain radical containing up to twenty fourcarbon atoms and having at least one carbon-carbon double bond. Examplesof alkenyl groups include, but are not limited to, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and thelike. Alkenyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkenyl groups as used herein mayoptionally include one or more further substituent groups.

As used herein, the term “alkynyl,” as used herein, refers to a straightor branched hydrocarbon radical containing up to twenty four carbonatoms and having at least one carbon-carbon triple bond. Examples ofalkynyl groups include, but are not limited to, ethynyl, 1-propynyl,1-butynyl, and the like. Alkynyl groups typically include from 2 toabout 24 carbon atoms, more typically from 2 to about 12 carbon atomswith from 2 to about 6 carbon atoms being more preferred. Alkynyl groupsas used herein may optionally include one or more further substitutentgroups.

As used herein, the term “aminoalkyl” as used herein, refers to an aminosubstituted alkyl radical. This term is meant to include C1-C12 alkylgroups having an amino substituent at any position and wherein the alkylgroup attaches the aminoalkyl group to the parent molecule. The alkyland/or amino portions of the aminoalkyl group can be further substitutedwith substituent groups.

As used herein, the term “aliphatic,” as used herein, refers to astraight or branched hydrocarbon radical containing up to twenty fourcarbon atoms wherein the saturation between any two carbon atoms is asingle, double or triple bond. An aliphatic group preferably containsfrom 1 to about 24 carbon atoms, more typically from 1 to about 12carbon atoms with from 1 to about 6 carbon atoms being more preferred.The straight or branched chain of an aliphatic group may be interruptedwith one or more heteroatoms that include nitrogen, oxygen, sulfur andphosphorus. Such aliphatic groups interrupted by heteroatoms includewithout limitation polyalkoxys, such as polyalkylene glycols,polyamines, and polyimines. Aliphatic groups as used herein mayoptionally include further substitutent groups.

As used herein, the term “alicyclic” or “alicyclyl” refers to a cyclicring system wherein the ring is aliphatic. The ring system can compriseone or more rings wherein at least one ring is aliphatic. Preferredalicyclics include rings having from about 5 to about 9 carbon atoms inthe ring. Alicyclic as used herein may optionally include furthersubstitutent groups. As used herein, the term “alkoxy,” as used herein,refers to a radical formed between an alkyl group and an oxygen atomwherein the oxygen atom is used to attach the alkoxy group to a parentmolecule. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as usedherein may optionally include further substitutent groups. As usedherein, the terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As used herein, the terms “aryl” and “aromatic,” as used herein, referto a mono- or polycyclic carbocyclic ring system radicals having one ormore aromatic rings. Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl andthe like. Preferred aryl ring systems have from about 5 to about 20carbon atoms in one or more rings. Aryl groups as used herein mayoptionally include further substitutent groups.

As used herein, the terms “aralkyl” and “arylalkyl,” as used herein,refer to a radical formed between an alkyl group and an aryl groupwherein the alkyl group is used to attach the aralkyl group to a parentmolecule. Examples include, but are not limited to, benzyl, phenethyland the like. Aralkyl groups as used herein may optionally includefurther substitutent groups attached to the alkyl, the aryl or bothgroups that form the radical group.

As used herein, the term “heterocyclic radical” as used herein, refersto a radical mono-, or poly-cyclic ring system that includes at leastone heteroatom and is unsaturated, partially saturated or fullysaturated, thereby including heteroaryl groups. Heterocyclic is alsomeant to include fused ring systems wherein one or more of the fusedrings contain at least one heteroatom and the other rings can containone or more heteroatoms or optionally contain no heteroatoms. Aheterocyclic group typically includes at least one atom selected fromsulfur, nitrogen or oxygen. Examples of heterocyclic groups include,[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as usedherein may optionally include further substitutent groups. As usedherein, the terms “heteroaryl,” and “heteroaromatic,” as used herein,refer to a radical comprising a mono- or poly-cyclic aromatic ring, ringsystem or fused ring system wherein at least one of the rings isaromatic and includes one or more heteroatom. Heteroaryl is also meantto include fused ring systems including systems where one or more of thefused rings contain no heteroatoms. Heteroaryl groups typically includeone ring atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

As used herein, the term “heteroarylalkyl,” as used herein, refers to aheteroaryl group as previously defined having an alky radical that canattach the heteroarylalkyl group to a parent molecule. Examples include,but are not limited to, pyridinylmethyl, pyrimidinylethyl,napthyridinylpropyl and the like. Heteroarylalkyl groups as used hereinmay optionally include further substitutent groups on one or both of theheteroaryl or alkyl portions.

As used herein, the term “mono or poly cyclic structure” as used in thepresent invention includes all ring systems that are single orpolycyclic having rings that are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl. Such mono andpoly cyclic structures can contain rings that are uniform or havevarying degrees of saturation including fully saturated, partiallysaturated or fully unsaturated. Each ring can comprise ring atomsselected from C, N, O and S to give rise to heterocyclic rings as wellas rings comprising only C ring atoms which can be present in a mixedmotif such as for example benzimidazole wherein one ring has only carbonring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic structures can be further substituted with substituent groupssuch as for example phthalimide which has two ═O groups attached to oneof the rings. In another aspect, mono or poly cyclic structures can beattached to a parent molecule directly through a ring atom, through asubstituent group or a bifunctional linking moiety.

As used herein, the term “acyl,” as used herein, refers to a radicalformed by removal of a hydroxyl group from an organic acid and has thegeneral formula C(O)—X where X is typically aliphatic, alicyclic oraromatic. Examples include aliphatic carbonyls, aromatic carbonyls,aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromaticphosphates, aliphatic phosphates and the like. Acyl groups as usedherein may optionally include further substitutent groups.

As used herein, the term “hydrocarbyl” includes groups comprising C, Oand H. Included are straight, branched and cyclic groups having anydegree of saturation. Such hydrocarbyl groups can include one or moreheteroatoms selected from N, O and S and can be further mono or polysubstituted with one or more substituent groups.

As used herein, the terms “substituent” and “substituent group,” as usedherein, include groups that are typically added to other groups orparent compounds to enhance desired properties or give desired effects.Substituent groups can be protected or unprotected and can be added toone available site or to many available sites in a parent compound.Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound. Such groupsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclicgroups, alkoxy, substituted oxo (—O—Raa), aryl, aralkyl, heterocyclic,heteroaryl, heteroarylalkyl, amino (—NRbbRcc), imino (═NRbb), amido(—C(O)N—RbbRcc or N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano(—CN), carbamido (—OC(O)NRbbRcc or N(Rbb)C(O)ORaa), ureido(—N(Rbb)C(O)NRbbRcc), thioureido (—N(Rbb)C(S)NRbbRcc), guanidinyl(—N(Rbb)C(═NRbb)NRbbRcc), amidinyl (—C(═NRbb)-NRbbRcc orN(Rbb)C(NRbb)Raa), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl(—S(O)2Rbb), sulfonamidyl (—S(O)2NRbbRcc or N(Rbb)S(O)2Rbb) andconjugate groups. Wherein each Raa, Rbb and Rcc is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation H, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl.

The REVERSIR compounds disclosed herein are particularly effective inreducing the activity of siRNAs. For example, the REVERSIR compoundsdisclosed herein can reduce the activity of an siRNA by at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90%, or at least about 95%, or at least about97%, or at least about 99% or up to and including a 100% decrease (i.e.,absent level as compared to a reference sample), or any decrease between50-100% as compared to a reference level. The reference level can besiRNA activity in absence of the REVERSIR compound.

In some embodiments, the REVERSIR compounds describe herein can reducethe activity of the siRNA by at least 75%, for example by 80%, 85%, 90%,95% or more and upto and including completer reduction or inhibition ofsiRNA activity, within less than seven (e.g., within 6 days, five days,four days, three days, two days or one day) of administering or use ofthe REVERSIR compound.

In some embodiments, the REVERSIR compounds can completely reduce thesiRNA activity within four days of administering or use of the REVERSIRcompound. By complete reduction of siRNA activity is meant a reductionof the siRNA activity by at least 80% relative to a reference level.

Oligomeric Compounds

In certain embodiments, the siRNA and/or the REVERSIR compounds areoligomeric compounds. In certain embodiments, it is desirable tochemically modify oligomeric compounds, including siRNAs and/or REVERSIRcompounds, compared to naturally occurring oligomers, such as DNA orRNA. Certain such modifications alter the activity of the oligomericcompound. Certain such chemical modifications can alter activity by, forexample: increasing affinity of a siRNA for its target nucleic acid or aREVERSIR for its target siRNA, increasing its resistance to one or morenucleases, and/or altering the pharmacokinetics or tissue distributionof the oligomeric compound. In certain instances, the use of chemistriesthat increase the affinity of an oligomeric compound for its target canallow for the use of shorter oligomeric compounds.

Monomers

In certain embodiment, oligomeric compounds comprise one or moremodified monomer. In certain such embodiments, oligomeric compoundscomprise one or more high affinity monomer. In certain embodiments, suchhigh-affinity monomer is selected from monomers (e.g., nucleosides andnucleotides) comprising 2′-modified sugars, including, but not limitedto: BNA's and monomers (e.g., nucleosides and nucleotides) with2′-substituents such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, OCF₃, O—(CH₂)₂—O—CH3, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(Rm)(Rn), orO—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, the oligomeric compounds including, but notlimited to REVERSIR compounds and siRNAs of the present invention,comprise one or more high affinity monomers.

In certain embodiments, the oligomeric compounds including, but notlimited to REVERSIR compounds and siRNAs of the present invention,comprise one or more 3-D-Methyleneoxy (4′-CH₂—O-2′) LNA monomers.

In certain embodiments, the oligomeric compounds including, including,but not limited to REVERSIR compounds and siRNAs of the presentinvention, comprise one or more α-D-Methyleneoxy (4′-CH₂—O-2′) LNAmonomers.

In certain embodiments, the oligomeric compounds including, including,but not limited to REVERSIR compounds and siRNAs of the presentinvention, comprise one or more (S)-cEt monomers.

In certain embodiments, the oligomeric compounds including, but notlimited to REVERSIR compounds and siRNAs of the present invention,comprise one or more high affinity monomers provided that the oligomericcompound does not comprise a nucleotide comprising a 2′-O(CH2)_(n)H,wherein n is one to six.

In certain embodiments, the oligomeric compounds including, but notlimited to REVERSIR compounds and siRNAs, comprise one or more highaffinity monomer provided that the oligomeric compound does not comprisea nucleotide comprising a 2′-OCH₃ or a 2′-O(CH₂)₂OCH₃.

In certain embodiments, the oligomeric compounds including, but notlimited to REVERSIR compounds and siRNAs, comprise one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) high affinitymonomer provided that the oligomeric compound does not comprise aα-L-Methyleneoxy (4′-CH₂—O-2′) LNA.

In certain embodiments, the oligomeric compounds including, but nolimited to REVERSIR compounds and siRNAs, comprise one or more highaffinity monomer provided that the oligomeric compound does not comprisea β-D-Methyleneoxy (4′-CH₂—O-2′) LNA.

In certain embodiments, the oligomeric compounds including, but nolimited to REVERSIR compound and siRNAs, comprise one or more highaffinity monomer provided that the oligomeric compound does not comprisea α-L-Methyleneoxy (4′-CH₂—O-2′) LNA or β-D-Methyleneoxy (4′-CH₂—O-2′)LNA.

Certain Nucleobases

The naturally occurring base portion of a nucleoside is typically aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. For those nucleosides thatinclude a pentofuranosyl sugar, a phosphate group can be linked to the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,those phosphate groups covalently link adjacent nucleosides to oneanother to form a linear polymeric compound. Within oligonucleotides,the phosphate groups are commonly referred to as forming theinternucleoside backbone of the oligonucleotide. The naturally occurringlinkage or backbone of RNA and of DNA is a 3′ to 5′ phosphodiesterlinkage.

In addition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable withthe compounds described herein. The unmodified or natural nucleobasescan be modified or replaced to provide oligonucleotides having improvedproperties. For example, nuclease resistant oligonucleotides can beprepared with these bases or with synthetic and natural nucleobases(e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, ortubercidine) and any one of the oligomer modifications described herein.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. When a natural base is replacedby a non-natural and/or universal base, the nucleotide is said tocomprise a modified nucleobase and/or a nucleobase modification herein.Modified nucleobase and/or nucleobase modifications also includenatural, non-natural and universal bases, which comprise conjugatedmoieties, e.g. a ligand described herein. Preferred conjugate moietiesfor conjugation with nucleobases include cationic amino groups which canbe conjugated to the nucleobase via an appropriate alkyl, alkenyl or alinker with an amide linkage.

An oligomeric compound described herein can also include nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Exemplary modifiednucleobases include, but are not limited to, other synthetic and naturalnucleobases such as inosine, xanthine, hypoxanthine, nubularine,isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine,2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine,2-(aminopropyl)adenine, 2-(methylthio)-N⁶-(isopentenyl)adenine,6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine,8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine,8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine,8-(thioalkyl)adenine, 8-(thiol)adenine, N⁶-(isopentyl)adenine,N⁶-(methyl)adenine, N⁶, N⁶-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine,7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine,8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine,8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine,8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine,3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine,5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine,5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine,5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N⁴-(acetyl)cytosine,3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil,4-(thio)uracil, 5-(methyl)-4-(thio)uracil,5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil,5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil,5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil,5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil,5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil,5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid,5-(methoxycarbonylmethyl)-2-(thio)uracil,5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,dihydrouracil, N³-(methyl)uracil, 5-uracil (i.e., pseudouracil),2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil,5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil,5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil,5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil,1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil,1-substituted 4-(thio)pseudouracil, 1-substituted2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil,1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl,3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, difluorotolyl,4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substitutedpyrimidines, N²-substituted purines, N⁶-substituted purines,O⁶-substituted purines, substituted 1,2,4-triazoles,pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylatedderivatives thereof. Alternatively, substituted or modified analogs ofany of the above bases and “universal bases” can be employed.

As used herein, a universal nucleobase is any nucleobase that can basepair with all of the four naturally occurring nucleobases withoutsubstantially affecting the melting behavior, recognition byintracellular enzymes or activity of the oligonucleotide duplex. Someexemplary universal nucleobases include, but are not limited to,2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine,4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methylisocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, and structural derivatives thereof (see forexample, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808;those disclosed in International Application No. PCT/US09/038425, filedMar. 26, 2009; those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990; those disclosed by English et al., AngewandteChemie, International Edition, 1991, 30, 613; those disclosed inModified Nucleosides in Biochemistry, Biotechnology and Medicine,Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Eds., CRC Press, 1993. Contents of all of theabove are herein incorporated by reference.

In certain embodiments, a modified nucleobase is a nucleobase that isfairly similar in structure to the parent nucleobase, such as forexample a 7-deaza purine, a 5-methyl cytosine, or a G-clamp. In certainembodiments, nucleobase mimetic include more complicated structures,such as for example a tricyclic phenoxazine nucleobase mimetic. Methodsfor preparation of the above noted modified nucleobases are well knownto those skilled in the art.

In some embodiments, the REVERSIR compound comprises at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) G-clampnucleobase selected from the following:

where n is 0, 1, 2, 3, 4, 5 or 6.

Certain Sugars

Oligomeric compounds provided herein can comprise one or more monomer,including a nucleoside or nucleotide, having a modified sugar moiety.For example, the furanosyl sugar ring of a nucleoside can be modified ina number of ways including, but not limited to, addition of asubstituent group, bridging of two non-geminal ring atoms to form alocked nucleic acid or bicyclic nucleic acid. In certain embodiments,oligomeric compounds comprise one or more monomers that are LNA.

In some embodiments of a locked nucleic acid, the 2′ position offurnaosyl is connected to the 4′ position by a linker selectedindependently from —[C(R1)(R2)]_(n)-, —[C(R1)(R2)]_(n)-O—,—[C(R1)(R2)]_(n)-N(R1)-, —[C(R1)(R2)]_(n)-N(R1)-O—,[C(R1R2)]_(n)-O—N(R1)-, —C(R1)=C(R2)-O—, —C(R1)=N—, —C(R1)=N—O—,C(═NR1)-, C(═NR1)-O—, C(═O)—, C(═O)O—, C(═S)—, C(═S)O—, C(═S)S—, O,Si(R1)2-, S(═O)_(x)— and N(R1)-;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R1 and R2 is, independently, H, a protecting group, hydroxyl,C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substitutedC2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,substituted C5-C20 aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical,substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3,COOJ1, acyl (C(═O) H), substituted acyl, CN, sulfonyl (S(═O)2-J1), orsulfoxyl (S(═O)-J1); and

each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl,substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl ora protecting group.

In one embodiment, each of the linkers of the LNA compounds is,independently, [C(R1)(R2)]_(n)-, [C(R1)(R2)]_(n)-O—, C(R1R2)-N(R1)-O orC(R1R2)-O—N(R1)-. In another embodiment, each of said linkers is,independently, 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R1)-2′ and 4′-CH₂—N(R1)-O-2′- wherein each R1is, independently, H, a protecting group or C1-C12 alkyl.

Certain LNA's have been prepared and disclosed in the patent literatureas well as in scientific literature (Singh et al., Chem. Commun., 1998,4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedtet al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Examples of issued US patents and published applications that discloseLNA s include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490;6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-GrantPublication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841;2004-0143114; and 20030082807.

Also provided herein are LNAs in which the 2′-hydroxyl group of theribosyl sugar ring is linked to the 4′ carbon atom of the sugar ringthereby forming a methyleneoxy (4′-CH₂—O-2′) linkage to form thebicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens.Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; andOrum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S.Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene(—CH₂—) group bridging the 2′ oxygen atom and the 4′ carbon atom, forwhich the term methyleneoxy (4′-CH₂—O-2′) LNA is used for the bicyclicmoiety; in the case of an ethylene group in this position, the termethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used (Singh et al., Chem. Commun.,1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003,11, 2211-2226). Methyleneoxy (4′-CH₂—O-2′) LNA and other bicyclic sugaranalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolyticdegradation and good solubility properties. Potent and nontoxicantisense oligonucleotides comprising BNAs have been described(Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

An isomer of methyleneoxy (4′-CH₂—O-2′) LNA that has also been discussedis alpha-L-methyleneoxy (4′-CH₂—O-2′) LNA which has been shown to havesuperior stability against a 3′-exonuclease. The alpha-L-methyleneoxy(4′-CH₂—O-2′) LNA's were incorporated into antisense gapmers andchimeras that showed potent antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) LNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) LNA, phosphorothioate-methyleneoxy(4′-CH₂—O-2′) LNA and 2′-thio-LNAs, have also been prepared (Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation oflocked nucleoside analogs comprising oligodeoxyribonucleotide duplexesas substrates for nucleic acid polymerases has also been described(Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-LNA, anovel comformationally restricted high-affinity oligonucleotide analoghas been described in the art (Singh et al., J. Org. Chem., 1998, 63,10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have beenprepared and the thermal stability of their duplexes with complementaryRNA and DNA strands has been previously reported.

Modified sugar moieties are well known and can be used to alter,typically increase, the affinity of the antisense compound for itstarget and/or increase nuclease resistance. A representative list ofpreferred modified sugars includes but is not limited to bicyclicmodified sugars, including methyleneoxy (4′-CH₂—O-2′) LNA andethyleneoxy (4′-(CH₂)₂—O-2′ bridge) ENA; substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH₃ or a 2′-O(CH₂)₂—OCH₃substituent group; and 4′-thio modified sugars. Sugars can also bereplaced with sugar mimetic groups among others. Methods for thepreparations of modified sugars are well known to those skilled in theart. Some representative patents and publications that teach thepreparation of such modified sugars include, but are not limited to,U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032;and WO 2005/121371.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR, n=1-50;“locked” nucleic acids (LNA) in which the furanose portion of thenucleoside includes a bridge connecting two carbon atoms on the furanosering, thereby forming a bicyclic ring system; O-AMINE orO—(CH₂)_(n)AMINE (n=1-10, AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, ethylene diamine or polyamino); and O—CH₂CH₂(NCH₂CH₂NMe₂)₂.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the single-strand overhangs); halo (e.g.,fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino); —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy;thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which can beoptionally substituted with e.g., an amino functionality.

Other suitable 2′-modifications, e.g., modified MOE, are described inU.S. Patent Application Publication No. 20130130378, contents of whichare herein incorporated by reference.

A modification at the 2′ position can be present in the arabinoseconfiguration The term “arabinose configuration” refers to the placementof a substituent on the C2′ of ribose in the same configuration as the2′-OH is in the arabinose.

The sugar can comprise two different modifications at the same carbon inthe sugar, e.g., gem modification. The sugar group can also contain oneor more carbons that possess the opposite stereochemical configurationthan that of the corresponding carbon in ribose. Thus, an oligomericcompound can include one or more monomers containing e.g., arabinose, asthe sugar. The monomer can have an alpha linkage at the 1′ position onthe sugar, e.g., alpha-nucleosides. The monomer can also have theopposite configuration at the 4′-position, e.g., C5′ and H4′ orsubstituents replacing them are interchanged with each other. When theC5′ and H4′ or substituents replacing them are interchanged with eachother, the sugar is said to be modified at the 4′ position.

Oligomeric compounds can also include abasic sugars, i.e., a sugar whichlack a nucleobase at C-1′ or has other chemical groups in place of anucleobase at C1′. See for example U.S. Pat. No. 5,998,203, content ofwhich is herein incorporated in its entirety. These abasic sugars canalso be further containing modifications at one or more of theconstituent sugar atoms. Oligomeric compounds can also contain one ormore sugars that are the L isomer, e.g. L-nucleosides. Modification tothe sugar group can also include replacement of the 4′-O with a sulfur,optionally substituted nitrogen or CH2 group. In some embodiments,linkage between C1′ and nucleobase is in a configuration.

Sugar modifications can also include acyclic nucleotides, wherein a C—Cbonds between ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′,C1′-O4′) is absent and/or at least one of ribose carbons or oxygen(e.g., C1′, C2′, C3′, C4′ or O4′) are independently or in combinationabsent from the nucleotide. In some embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R₁ and R₂independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar).

In some embodiments, sugar modifications are selected from the groupconsisting of 2′-H, 2′-O-Me (2′-O-methyl), 2′-O-MOE (2′-O-methoxyethyl),2′-F, 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), 2′-S-methyl,2′-O—CH₂-(4′-C) (LNA), 2′-O—CH₂CH₂-(4′-C) (ENA), 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE) and gem 2′-OMe/2′F with 2′-O-Me in the arabinoseconfiguration.

It is to be understood that when a particular nucleotide is linkedthrough its 2′-position to the next nucleotide, the sugar modificationsdescribed herein can be placed at the 3′-position of the sugar for thatparticular nucleotide, e.g., the nucleotide that is linked through its2′-position. A modification at the 3′ position can be present in thexylose configuration The term “xylose configuration” refers to theplacement of a substituent on the C3′ of ribose in the sameconfiguration as the 3′-OH is in the xylose sugar.

The hydrogen attached to C4′ and/or C1′ can be replaced by a straight-or branched-optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, wherein backbone of the alkyl,alkenyl and alkynyl can contain one or more of O, S, S(O), SO₂, N(R′),C(O), N(R′)C(O)O, OC(O)N(R′), CH(Z′), phosphorous containing linkage,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclic or optionally substitutedcycloalkyl, where R′ is hydrogen, acyl or optionally substitutedaliphatic, Z′ is selected from the group consisting of OR₁₁, COR₁₁,CO₂R₁₁,

NR₂₁R₃₁, CONR₂₁R₃₁, CON(H)NR₂₁R₃₁, ONR₂₁R₃₁, CON(H)N═CR₄₁R₅₁,N(R₂₁)C(═NR₃₁)NR₂₁R₃₁, N(R₂₁)C(O)NR₂₁R₃₁, N(R₂₁)C(S)NR₂₁R₃₁,OC(O)NR₂₁R₃₁, SC(O)NR₂₁R₃₁, N(R₂₁)C(S)OR₁₁, N(R₂₁)C(O)OR₁₁,N(R₂₁)C(O)SR₁₁, N(R₂₁)N═CR₄₁R₅₁, ON═CR₄₁R₅₁, SO₂R₁₁, SOR₁₁, SR₁₁, andsubstituted or unsubstituted heterocyclic; R₂₁ and R₃₁ for eachoccurrence are independently hydrogen, acyl, unsubstituted orsubstituted aliphatic, aryl heteroaryl, heterocyclic, OR₁₁, COR₁₁,CO₂R₁₁, or NR₁₁R₁₁′; or R₂₁ and R₃₁, taken together with the atoms towhich they are attached, form a heterocyclic ring; R₄₁ and R₅₁ for eachoccurrence are independently hydrogen, acyl, unsubstituted orsubstituted aliphatic, aryl, heteroaryl, heterocyclic, OR₁₁, COR₁₁, orCO₂R₁₁, or NR₁₁R₁₁′; and R₁₁ and R₁₁′ are independently hydrogen,aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. Insome embodiments, the hydrogen attached to the C4′ of the 5′ terminalnucleotide is replaced.

In some embodiments, C4′ and C5′ together form an optionally substitutedheterocyclic, preferably comprising at least one —PX(Y)—, wherein X isH, OH, OM, SH, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkylthio, optionally substitutedalkylamino or optionally substituted dialkylamino, where M isindependently for each occurrence an alki metal or transition metal withan overall charge of +1; and Y is O, S, or NR′, where R′ is hydrogen,optionally substituted aliphatic. Preferably this modification is at the5 terminal of the oligonucleotide.

In certain embodiments, LNA's include bicyclic nucleoside having theformula:

wherein:

-   -   Bx is a heterocyclic base moiety;    -   T1 is H or a hydroxyl protecting group;    -   T2 is H, a hydroxyl protecting group or a reactive phosphorus        group;    -   Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted        C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6        alkynyl, acyl, substituted acyl, or substituted amide.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with optionally protected substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3,OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.

In certain such embodiments, each of the substituted groups, is,independently, mono or poly substituted with substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3,OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is,independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O orNJ 1.

In certain embodiments, the Z group is C1-C6 alkyl substituted with oneor more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3,OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In anotherembodiment, the Z group is C1-C6 alkyl substituted with one or more Xx,wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy(e.g., CH3O—), substituted alkoxy or azido.

In certain embodiments, the Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2,SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. Inanother embodiment, the Z group is —CH2Xx, wherein Xx is halo (e.g.,fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

In certain such embodiments, the Z group is in the (R)-configuration:

In certain such embodiments, the Z group is in the (S)-configuration:

In certain embodiments, each T1 and T2 is a hydroxyl protecting group. Apreferred list of hydroxyl protecting groups includes benzyl, benzoyl,2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T1is a hydroxyl protecting group selected from acetyl, benzyl,t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein amore preferred hydroxyl protecting group is T1 is 4,4′-dimethoxytrityl.

In certain embodiments, T2 is a reactive phosphorus group whereinpreferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments T1 is4,4′-dimethoxytrityl and T2 is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, oligomeric compounds have at least one monomerof the formula:

or of the formula:

or of the formula:

wherein

-   -   Bx is a heterocyclic base moiety;    -   T3 is H, a hydroxyl protecting group, a linked conjugate group        or an internucleoside linking group attached to a nucleoside, a        nucleotide, an oligonucleoside, an oligonucleotide, a monomeric        subunit or an oligomeric compound;    -   T4 is H, a hydroxyl protecting group, a linked conjugate group        or an internucleoside linking group attached to a nucleoside, a        nucleotide, an oligonucleoside, an oligonucleotide, a monomeric        subunit or an oligomeric compound;    -   wherein at least one of T3 and T4 is an internucleoside linking        group attached to a nucleoside, a nucleotide, an        oligonucleoside, an oligonucleotide, a monomeric subunit or an        oligomeric compound; and    -   Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted        C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6        alkynyl, acyl, substituted acyl, or substituted amide.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with optionally protected substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3,OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, andNJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H or C1-C6alkyl, and X is O or NJ1.

In certain such embodiments, at least one Z is C1-C6 alkyl orsubstituted C1-C6 alkyl. In certain embodiments, each Z is,independently, C1-C6 alkyl or substituted C1-C6 alkyl. In certainembodiments, at least one Z is C1-C6 alkyl. In certain embodiments, eachZ is, independently, C1-C6 alkyl. In certain embodiments, at least one Zis methyl. In certain embodiments, each Z is methyl. In certainembodiments, at least one Z is ethyl. In certain embodiments, each Z isethyl. In certain embodiments, at least one Z is substituted C1-C6alkyl. In certain embodiments, each Z is, independently, substitutedC1-C6 alkyl. In certain embodiments, at least one Z is substitutedmethyl. In certain embodiments, each Z is substituted methyl. In certainembodiments, at least one Z is substituted ethyl. In certainembodiments, each Z is substituted ethyl.

In certain embodiments, at least one substituent group is C1-C6 alkoxy(e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6alkoxy). In another embodiment, each substituent group is,independently, C1-C6 alkoxy (e.g., each Z is, independently, C1-C6 alkylsubstituted with one or more C1-C6 alkoxy).

In certain embodiments, at least one C1-C6 alkoxy substituent group isCH3O— (e.g., at least one Z is CH₃OCH₂—). In another embodiment, eachC1-C6 alkoxy substituent group is CH₃O— (e.g., each Z is CH₃OCH₂—).

In certain embodiments, at least one substituent group is halogen (e.g.,at least one Z is C1-C6 alkyl substituted with one or more halogen). Incertain embodiments, each substituent group is, independently, halogen(e.g., each Z is, independently, C1-C6 alkyl substituted with one ormore halogen). In certain embodiments, at least one halogen substituentgroup is fluoro (e.g., at least one Z is CH₂FCH₂—, CHF₂CH₂— or CF₃CH₂—).In certain embodiments, each halo substituent group is fluoro (e.g.,each Z is, independently, CH₂FCH₂—, CHF₂CH₂— or CF₃CH₂—).

In certain embodiments, at least one substituent group is hydroxyl(e.g., at least one Z is C1-C6 alkyl substituted with one or morehydroxyl). In certain embodiments, each substituent group is,independently, hydroxyl (e.g., each Z is, independently, C1-C6 alkylsubstituted with one or more hydroxyl). In certain embodiments, at leastone Z is HOCH₂—. In another embodiment, each Z is HOCH₂—.

In certain embodiments, at least one Z is CH₃—, CH₃CH₂—, CH₂OCH₃—, CH₂F—or HOCH₂—. In certain embodiments, each Z is, independently, CH₃—,CH₃CH₂—, CH₂OCH₃—, CH₂F— or HOCH₂—.

In certain embodiments, at least one Z group is C1-C6 alkyl substitutedwith one or more Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1,N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 andJ3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In anotherembodiment, at least one Z group is C1-C6 alkyl substituted with one ormore Xx, wherein each Xx is, independently, halo (e.g., fluoro),hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain embodiments, each Z group is, independently, C1-C6 alkylsubstituted with one or more Xx, wherein each Xx is independently OJ1,NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein eachJ1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.In another embodiment, each Z group is, independently, C1-C6 alkylsubstituted with one or more Xx, wherein each Xx is independently halo(e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain embodiments, at least one Z group is —CH₂Xx, wherein Xx isOJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; whereineach J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S orNJ1 In certain embodiments, at least one Z group is —CH₂Xx, wherein Xxis halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain embodiments, each Z group is, independently, —CH₂Xx, whereineach Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2,NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H orC1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z groupis, independently, —CH₂Xx, wherein each Xx is, independently, halo(e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain embodiments, at least one Z is CH₃—. In another embodiment,each Z is, CH₃—.

In certain embodiments, the Z group of at least one monomer is in the(R)-configuration represented by the formula:

or the formula:

or the formula:

IN certain embodiments, the Z group of each monomer of the formula is inthe (R)-configuration.

In certain embodiments, the Z group of at least one monomer is in the(S)-configuration represented by the formula:

or the formula:

or the formula:

In certain embodiments, the Z group of each monomer of the formula is inthe (S)-configuration.

In certain embodiments, T3 is H or a hydroxyl protecting group. Incertain embodiments, T4 is H or a hydroxyl protecting group. In afurther embodiment T3 is an internucleoside linking group attached to anucleoside, a nucleotide or a monomeric subunit. In certain embodiments,T4 is an internucleoside linking group attached to a nucleoside, anucleotide or a monomeric subunit. In certain embodiments, T3 is aninternucleoside linking group attached to an oligonucleoside or anoligonucleotide. In certain embodiments, T4 is an internucleosidelinking group attached to an oligonucleoside or an oligonucleotide. Incertain embodiments, T3 is an internucleoside linking group attached toan oligomeric compound. In certain embodiments, T4 is an internucleosidelinking group attached to an oligomeric compound. In certainembodiments, at least one of T3 and T4 comprises an internucleosidelinking group selected from phosphodiester or phosphorothioate.

In certain embodiments, oligomeric compounds have at least one region ofat least two contiguous monomers of the formula:

or of the formula:

or of the formula:

In certain such embodiments, LNAs include, but are not limited to, (A)α-L-Methyleneoxy (4′-CH2-O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH2-O-2′)LNA, (C) Ethyleneoxy (4′-(CH2)2-O-2′) LNA, (D) Aminooxy(4′-CH2-O—N(R)-2′) LNA and (E) Oxyamino (4′-CH2-N(R)—O-2′) LNA, asdepicted below:

In certain embodiments, the oligomeric compound comprises at least tworegions of at least two contiguous monomers of the above formula. Incertain embodiments, the oligomeric compound comprises a gappedoligomeric compound. In certain embodiments, the oligomeric compoundcomprises at least one region of from about 8 to about 14 contiguousβ-D-2′-deoxyribofuranosyl nucleosides. In certain embodiments, theoligomeric compound comprises at least one region of from about 9 toabout 12 contiguous β-D-2′-deoxyribofuranosyl nucleosides.

In certain embodiments, the oligomeric compound comprises at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more)S-cEtmonomer of the formula:

wherein Bx IS heterocyclic base moiety.

In some embodiments, the oligomeric compound, e.g. REVERSIR compound,comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or more) nucleoside selected from the following:

where B is A-001 to A-026 and n is 0-6 (e.g., 0, 1, 2, 3, 4, 5 or 6).

In certain embodiments, monomers include sugar mimetics. In certain suchembodiments, a mimetic is used in place of the sugar orsugar-internucleoside linkage combination, and the nucleobase ismaintained for hybridization to a selected target. Representativeexamples of a sugar mimetics include, but are not limited to,cyclohexenyl or morpholino. Representative examples of a mimetic for asugar-internucleoside linkage combination include, but are not limitedto, peptide nucleic acids (PNA) and morpholino groups linked byuncharged achiral linkages. In some instances a mimetic is used in placeof the nucleobase. Representative nucleobase mimetics are well known inthe art and include, but are not limited to, tricyclic phenoxazineanalogs and universal bases (Berger et al., Nuc Acid Res. 2000,28:2911-14, incorporated herein by reference). Methods of synthesis ofsugar, nucleoside and nucleobase mimetics are well known to thoseskilled in the art.

In certain embodiments, the REVERSIR compound comprises at least onemonomer that is LNA and at least one G-clamp nucleobase. For example,the REVERSIR compound can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more monomers that are LNA 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more G-clamp nucleobases.

In some embodiments, the REVERSIR compound comprises at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) peptidenucleic acid monomer. In certain embodiments, the REVERSIR compoundcomprises at least one monomer that is LNA and at least one monomer thatis PNA. For example, the REVERSIR compound can comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers that are LNA 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more monomers that arePNA.

In certain embodiments, the REVERSIR compound comprises at least one PNAmonomer and at least one G-clamp nucleobase. For example, the REVERSIRcompound can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more PNA monomers and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or more G-clamp nucleobases.

In certain embodiments, the REVERSIR compound comprises at least one LNAmonomer, at least one PNA monomer and at least one G-clamp nucleobase.For example, the REVERSIR compound can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more LNA monomers; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more PNA monomers and 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more G-clamp nucleobases.

Monomeric Linkages

Described herein are linking groups that link monomers (including, butnot limited to, modified and unmodified nucleosides and nucleotides)together, thereby forming an oligomeric compound. Such linking groupsare also referred to as intersugar linkage. The two main classes oflinking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing linkages include, but are notlimited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing linking groups include, but are not limitedto, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—),thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); andN,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-). Oligomeric compoundshaving non-phosphorus linking groups are referred to asoligonucleosides. Modified linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the oligomeric compound. In certain embodiments, linkages having achiral atom can be prepared a racemic mixtures, as separate enantomers.Representative chiral linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

The phosphate group in the linking group can be modified by replacingone of the oxygens with a different substituent. One result of thismodification can be increased resistance of the oligonucleotide tonucleolytic breakdown. Examples of modified phosphate groups includephosphorothioate, phosphoroselenates, borano phosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl oraryl phosphonates and phosphotriesters. In some embodiments, one of thenon-bridging phosphate oxygen atoms in the linkage can be replaced byany of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl), C (i.e.an alkyl group, an aryl group, etc. . . . ), H, NR₂ (R is hydrogen,optionally substituted alkyl, aryl), or OR (R is optionally substitutedalkyl or aryl). The phosphorous atom in an unmodified phosphate group isachiral. However, replacement of one of the non-bridging oxygens withone of the above atoms or groups of atoms renders the phosphorous atomchiral; in other words a phosphorous atom in a phosphate group modifiedin this way is a stereogenic center. The stereogenic phosphorous atomcan possess either the “R” configuration (herein Rp) or the “S”configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligonucleotides diastereomers. Thus, whilenot wishing to be bound by theory, modifications to both non-bridgingoxygens, which eliminate the chiral center, e.g. phosphorodithioateformation, can be desirable in that they cannot produce diastereomermixtures. Thus, the non-bridging oxygens can be independently any one ofO, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the sugar of themonomer), with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at the either one of the linking oxygens or atboth linking oxygens. When the bridging oxygen is the 3′-oxygen of anucleoside, replacement with carbon is preferred. When the bridgingoxygen is the 5′-oxygen of a nucleoside, replacement with nitrogen ispreferred.

Modified phosphate linkages where at least one of the oxygen linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non-phosphodiesterintersugar linkage” or “non-phosphodiester linker.”

In certain embodiments, the phosphate group can be replaced bynon-phosphorus containing connectors, e.g. dephospho linkers. Dephospholinkers are also referred to as non-phosphodiester linkers herein. Whilenot wishing to be bound by theory, it is believed that since the chargedphosphodiester group is the reaction center in nucleolytic degradation,its replacement with neutral structural mimics should impart enhancednuclease stability. Again, while not wishing to be bound by theory, itcan be desirable, in some embodiment, to introduce alterations in whichthe charged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include, butare not limited to, amides (for example amide-3 (3′-CH₂—C(═O)—N(H)-5′)and amide-4 (3′-CH₂—N(H)—C(═O)-5′)), hydroxylamino, siloxane(dialkylsiloxxane), carboxamide, carbonate, carboxymethyl, carbamate,carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate,sulfonamide, sulfonate ester, thioformacetal (3′-S—CH₂—O-5′), formacetal(3′-O—CH₂—O-5′), oxime, methyleneimino, methykenecarbonylamino,methylenemethylimino (MMI, 3′-CH₂—N(CH₃)—O-5′), methylenehydrazo,methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3′-O—C5′),thioethers (C3′-S—C5′), thioacetamido (C3′-N(H)—C(═O)—CH₂—S—C5′,C3′-O—P(O)—O—SS—C5′, C3′-CH₂—NH—NH—C5′, 3′-NHP(O)(OCH₃)—O-5′ and3′-NHP(O)(OCH₃)—O-5′ and nonionic linkages containing mixed N, O, S andCH₂ component parts. See for example, Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS SymposiumSeries 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments includemethylenemethylimino (MMI), methylenecarbonylamino, amides, carbamateand ethylene oxide linker.

One skilled in the art is well aware that in certain instancesreplacement of a non-bridging oxygen can lead to enhanced cleavage ofthe intersugar linkage by the neighboring 2′-OH, thus in many instances,a modification of a non-bridging oxygen can necessitate modification of2′-OH, e.g., a modification that does not participate in cleavage of theneighboring intersugar linkage, e.g., arabinose sugar, 2′-O-alkyl, 2′-F,LNA and ENA.

Preferred non-phosphodiester intersugar linkages includephosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric excess of Spisomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% 95% or more enantiomeric excess of Rp isomer,phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters,alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates,phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates.

In some embodiments, the oligomeric compound, e.g., REVERSIR compound orsiRNA, comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more and upto including all) modified ornonphosphodiester linkages. In one embodiment, the oligomeric compound,e.g., REVERSIR compound or siRNA, comprises at least one (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all)phosphorothioate linkages.

In some embodiments, all internucleoside linkages in the reversercompounds are phosphorothioate (PS) internucleoside linkages. In certainembodiments, the REVERSIR compounds comprise at least onephosphorothioate (PS) internucleoside linkage, but not allinternucleoside linkages in said REVERSIR compound are aphosphorothioate linkage. In other words, in some embodiments, less than100% (e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%or fewer) of the internucleoside linkages are phosphorothioate linkages.

In some embodiments, the REVERSIR compounds comprise at least onephosphorothioate internucleoside linkage and at least oneinternucleoside linkage that is not a phosphorothioate. For example, theREVERSIR compounds comprise at least one phosphorothioateinternucleoside linkage and at least one phosphodiester internucleosidelinkage. In some embodiments, the non-phosphorothioate internucleosidelinkage is between the terminus and the penultimate nucleosides.

In some embodiments, the internucleoside linkage between the nucleobaseat the 3′-terminus of the REVERSIR compound and the rest of the REVERSIRcompound is a phosphodiester linkage. In some embodiments, allinternucleoside linkages in the reverser compounds are phosphorothioateexcept for the internucleoside linkage between the nucleoside at the3′-terminus of the REVERSIR compound and the rest of the REVERSIRcompound.

Oligomeric compounds can also be constructed wherein the phosphatelinker and the sugar are replaced by nuclease resistant nucleoside ornucleotide surrogates. While not wishing to be bound by theory, it isbelieved that the absence of a repetitively charged backbone diminishesbinding to proteins that recognize polyanions (e.g. nucleases). Again,while not wishing to be bound by theory, it can be desirable in someembodiment, to introduce alterations in which the bases are tethered bya neutral surrogate backbone. Examples include the morpholino,cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycylPNA (aegPNA) and backnone-extended pyrrolidine PNA (bepPNA) nucleosidesurrogates. A preferred surrogate is a PNA surrogate.

The oligomeric compounds described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or(L) such as for amino acids et al. Included in the antisense compoundsprovided herein are all such possible isomers, as well as their racemicand optically pure forms.

Terminal Modifications

Ends of the oligomeric compound can be modified. Such modifications canbe at one end or both ends. For example, the 3′ and/or 5′ ends of anoligonucleotide can be conjugated to other functional molecular entitiessuch as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA,fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., onsulfur, silicon, boron or ester). The functional molecular entities canbe attached to the sugar through a phosphate group and/or a linker. Theterminal atom of the linker can connect to or replace the linking atomof the phosphate group or the C-3′ or C-5′ O, N, S or C group of thesugar. Alternatively, the linker can connect to or replace the terminalatom of a nucleotide surrogate (e.g., PNAs).

When a linker/phosphate-functional molecular entity-linker/phosphatearray is interposed between two strands of a double stranded oligomericcompound, this array can substitute for a hairpin loop in a hairpin-typeoligomeric compound.

Terminal modifications useful for modulating activity includemodification of the 5′ end of oligomeric compound with phosphate orphosphate analogs. In certain embodiments, the 5′end of oligomericcompound is phosphorylated or includes a phosphoryl analog. Exemplary5′-phosphate modifications include those which are compatible with RISCmediated gene silencing. Modifications at the 5′-terminal end can alsobe useful in stimulating or inhibiting the immune system of a subject.In some embodiments, the 5′-end of the oligomeric compound comprises themodification

wherein W, X and Y are each independently selected from the groupconsisting of 0, OR (R is hydrogen, alkyl, aryl), S, Se, BR₃ (R ishydrogen, alkyl, aryl), BH₃ ⁻, C (i.e. an alkyl group, an aryl group,etc. . . . ), H, NR₂ (R is hydrogen, alkyl, aryl), or OR (R is hydrogen,alkyl or aryl); A and Z are each independently for each occurrenceabsent, O, S, CH₂, NR (R is hydrogen, alkyl, aryl), or optionallysubstituted alkylene, wherein backbone of the alkylene can comprise oneor more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internallyand/or at the end; and n is 0-2. In some embodiments, n is 1 or 2. It isunderstood that A is replacing the oxygen linked to 5′ carbon of sugar.When n is 0, W and Y together with the P to which they are attached canform an optionally substituted 5-8 membered heterocyclic, wherein W an Yare each independently O, S, NR′ or alkylene. Preferably theheterocyclic is substituted with an aryl or heteroaryl. In someembodiments, one or both hydrogen on C5′ of the 5′-terminal nucleotidesare replaced with a halogen, e.g., F.

Exemplary 5′-modifications include, but are not limited to,5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)2(O)P—S-5′); 5′-alpha-thiotriphosphate; 5′-beta-thiotriphosphate;5′-gamma-thiotriphosphate; 5′-phosphoramidates ((HO)₂(O)P—NH-5′,(HO)(NH₂)(O)P—O-5′). Other 5′-modification include 5′-alkylphosphonates(R(OH)(O)P—O-5′, R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc. .. . ), 5′-alkyletherphosphonates (R(OH)(O)P—O-5′, R=alkylether, e.g.,methoxymethyl (CH2OMe), ethoxymethyl, etc. . . . ). Other exemplary5′-modifications include where Z is optionally substituted alkyl atleast once, e.g., ((HO)₂(X)P—O[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′,((HO)₂(X)P—O[—(CH₂)_(a)—P(X)(OH)—O]_(b)-5′,((HO)₂(X)P—[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′; dialkyl terminal phosphatesand phosphate mimics: HO[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′,H₂N[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′, H[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′,Me₂N[—(CH₂)_(a)—O—P(X)(OH)—O]_(b)-5′, HO[—(CH₂)_(a)—P(X)(OH)—O]_(b)-5′,H₂N[—(CH₂)_(a)—P(X)(OH)—O]_(b)-5′, H[—(CH₂)_(a)—P(X)(OH)—O]_(b)-5′,Me₂N[—(CH₂)_(a)—P(X)(OH)—O]_(b)-5′, wherein a and b are eachindependently 1-10. Other embodiments, include replacement of oxygenand/or sulfur with BH₃, BH₃ ⁻ and/or Se.

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include targeting ligands. Terminal modificationscan also be useful for cross-linking an oligonucleotide to anothermoiety; modifications useful for this include mitomycin C, psoralen, andderivatives thereof.

Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds havingreactive phosphorus groups useful for forming linkages including forexample phosphodiester and phosphorothioate internucleoside linkages.Methods of preparation and/or purification of precursors or oligomericcompounds are not a limitation of the compositions or methods providedherein. Methods for synthesis and purification of oligomeric compoundsincluding DNA, RNA, oligonucleotides, oligonucleosides, and antisensecompounds are well known to those skilled in the art.

Generally, oligomeric compounds comprise a plurality of monomericsubunits linked together by linking groups. Non-limiting examples ofoligomeric compounds include primers, probes, antisense compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, and siRNAs. As such, thesecompounds can be introduced in the form of single-stranded,double-stranded, circular, branched or hairpins and can containstructural elements such as internal or terminal bulges or loops.Oligomeric double-stranded compounds can be two strands hybridized toform double-stranded compounds or a single strand with sufficientself-complementarity to allow for hybridization and formation of a fullyor partially double-stranded compound.

In certain embodiments, the present invention provides chimericoligomeric compounds. In certain such embodiments, chimeric oligomericcompounds are chimeric oligonucleotides. In certain such embodiments,the chimeric oligonucleotides comprise differently modified nucleotides.In certain embodiments, chimeric oligonucleotides are mixed-backboneantisense oligonucleotides.

In general a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif. Any combination of modifications and/ormimetic groups can comprise a chimeric oligomeric compound as describedherein.

In certain embodiments, chimeric oligomeric compounds typically compriseat least one region modified so as to confer increased resistance tonuclease degradation, increased cellular uptake, and/or increasedbinding affinity for the target nucleic acid. In certain embodiments, anadditional region of the oligomeric compound may serve as a substratefor enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.

In certain embodiments, chimeric oligomeric compounds are gapmers. Incertain such embodiments, a mixed-backbone oligomeric compound has onetype of internucleotide linkages in one or both wings and a differenttype of internucleoside linkages in the gap. In certain suchembodiments, the mixed-backbone oligonucleotide has phosphodiesterlinkages in the wings and phosphorothioate linkages in the gap. Incertain embodiments in which the internucleoside linkages in a wing isdifferent from the internucleoside linkages in the gap, theinternucleoside linkage bridging that wing and the gap is the same asthe internucleoside linkage in the wing. In certain embodiments in whichthe internucleoside linkages in a wing is different from theinternucleoside linkages in the gap, the internucleoside linkagebridging that wing and the gap is the same as the internucleosidelinkage in the gap.

In certain embodiments, the present invention provides oligomericcompounds, including siRNAs and REVERSIR compounds of any of a varietyof ranges of lengths. In certain embodiments, the invention providesoligomeric compounds consisting of X—Y linked oligonucleosides, where Xand Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50;provided that X<Y. For example, in certain embodiments, the inventionprovides oligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12, 8-13,8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25,8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16,9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28,9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18,10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28,10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19,11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29,11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21,12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14,13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24,13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18,14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28,14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23,15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19,16-25, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29,16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26,17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24,18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23,19-24, 19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23,20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24,21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26,22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29,23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28,25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29,28-30, or 29-30 linked nucleosides.

As noted-above, REVERSIR compounds can be of any length. For example, insome embodiments, the REVERSIR compound is a modified oligonucleotideconsisting of 6-30 nucleotides. For example, the REVERSIR compound canconsist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleobases. In someembodiments, the REVERSIR compound consists of 6-17, 7-16 or 8-15 linkednucleobases.

The inventors have discovered inter alia that REVERSIR compounds, i.e.,modified oligonucleotides, consisting of 15 or fewer nucleosides areparticularly effective in reversing the siRNA activity. Accordingly, insome embodiments, the REVERSIR compound is a modified oligonucleotideconsisting of 8-15 (e.g., 8, 9, 10, 11, 12, 13, 14 or 15) linkednucleosides. In some embodiments, the REVERSIR compound is a modifiedoligonucleotide consisting of 6-12, 7-11 or 8-10 linked nucleobases. Insome embodiments, the REVERSIR compound consists of 8-9 linkednucleobases.

As discussed herein, REVERSIR compounds are modified oligonucleotidesthat are substantially complementary to at least one strand of an siRNA.Now without wishing to be bound by a theory, REVERSIR compounds that aresubstantially complementary to the seed region of the antisense strandof the siRNA (i.e., at positions 2-8 of the 5′-end of the antisensestrand) are particularly effective in reducing siRNA activity. Thus, inmany embodiments, the REVERSIR compound is substantially complementaryto nucleosides 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15 or 2-16 ofthe antisense strand of the siRNA. By substantially complementary inthis context is meant a complementarity of at least 90%, preferably atleast 95%, and more preferably complete complementarity.

Ligands

In certain embodiments, oligomeric compounds are modified by covalentattachment of one or more conjugate groups. In general, conjugate groupsmodify one or more properties of the attached oligomeric compoundincluding but not limited to pharmacodynamic, pharmacokinetic, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional linking moiety or linking groupto a parent compound such as an oligomeric compound. A preferred list ofconjugate groups includes without limitation, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, thioethers,polyethers, cholesterols, thiocholesterols, cholic acid moieties,folate, lipids, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarinsand dyes.

Preferred conjugate groups amenable to the present invention includelipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al.,Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g.,di-hexadecyl-rac-glycerol ortriethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264,229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety(Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Generally, a wide variety of entities, e.g., ligands, can be coupled tothe oligomeric compounds described herein. Ligands can include naturallyoccurring molecules, or recombinant or synthetic molecules. Exemplaryligands include, but are not limited to, polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K,PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamidepolymers, polyphosphazine, polyethylenimine, cationic groups, spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, mucin,glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate,polyaspartate, aptamer, asialofetuin, hyaluronan, procollagen,immunoglobulins (e.g., antibodies), insulin, transferrin, albumin,sugar-albumin conjugates, intercalating agents (e.g., acridines),cross-linkers (e.g. psoralen, mitomycin C), porphyrins (e.g., TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA),lipophilic molecules (e.g, steroids, bile acids, cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides(e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cellpermeation peptide, endosomolytic/fusogenic peptide), alkylating agents,phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl,radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., naproxen, aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies,hormones and hormone receptors, lectins, carbohydrates, multivalentcarbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitaminB, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamincofactors, lipopolysaccharide, an activator of p38 MAP kinase, anactivator of NF-κB, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, myoservin, tumor necrosis factor alpha (TNFalpha),interleukin-1 beta, gamma interferon, natural or recombinant low densitylipoprotein (LDL), natural or recombinant high-density lipoprotein(HDL), and a cell-permeation agent (e.g., a. helical cell-permeationagent).

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides. Apeptidomimetic (also referred to herein as an oligopeptidomimetic) is amolecule capable of folding into a defined three-dimensional structuresimilar to a natural peptide. The peptide or peptidomimetic ligand canbe about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35,40, 45, or 50 amino acids long.

Exemplary amphipathic peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides,esculentinis-1, and caerins.

As used herein, the term “endosomolytic ligand” refers to moleculeshaving endosomolytic properties. Endosomolytic ligands promote the lysisof and/or transport of the composition of the invention, or itscomponents, from the cellular compartments such as the endosome,lysosome, endoplasmic reticulum (ER), golgi apparatus, microtubule,peroxisome, or other vesicular bodies within the cell, to the cytoplasmof the cell. Some exemplary endosomolytic ligands include, but are notlimited to, imidazoles, poly or oligoimidazoles, linear or branchedpolyethyleneimines (PEIs), linear and brached polyamines, e.g. spermine,cationic linear and branched polyamines, polycarboxylates, polycations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketals, orthoesters, linear or branched polymers with maskedor unmasked cationic or anionic charges, dendrimers with masked orunmasked cationic or anionic charges, polyanionic peptides, polyanionicpeptidomimetics, pH-sensitive peptides, natural and synthetic fusogeniclipids, natural and synthetic cationic lipids.

Exemplary endosomolytic/fusogenic peptides include, but are not limitedto, AALEALAEALEALAEALEALAEAAAAGGC (GALA); AALAEALAEALAEALAEALAEALAAAAGGC(EALA); ALEALAEALEALAEA; GLFEAIEGFIENGWEGMIWDYG (INF-7);GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2);GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7);GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3);GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF);GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3); GLF EAI EGFI ENGW EGnIDG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine);LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTG1);GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA);GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin);H₅WYG; and CHK₆HC.

Without wishing to be bound by theory, fusogenic lipids fuse with andconsequently destabilize a membrane. Fusogenic lipids usually have smallhead groups and unsaturated acyl chains. Exemplary fusogenic lipidsinclude, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine(DOPE), phosphatidylethanolamine (POPE),palmitoyloleoylphosphatidylcholine (POPC),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin),N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine(DLin-k-DMA) andN-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine(also refered to as XTC herein).

Synthetic polymers with endosomolytic activity amenable to the presentinvention are described in U.S. Pat. App. Pub. Nos. 2009/0048410;2009/0023890; 2008/0287630; 2008/0287628; 2008/0281044; 2008/0281041;2008/0269450; 2007/0105804; 20070036865; and 2004/0198687, contents ofwhich are hereby incorporated by reference in their entirety.

Exemplary cell permeation peptides include, but are not limited to,RQIKIWFQNRRMKWKK (penetratin); GRKKRRQRRRPPQC (Tat fragment 48-60);GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based peptide);LLIILRRRIRKQAHAHSK (PVEC); GWTLNSAGYLLKINLKALAALAKKIL (transportan);KLALKLALKALKAALKLA (amphiphilic model peptide); RRRRRRRRR (Arg9);KFFKFFKFFK (Bacterial cell wall permeating peptide);LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37);SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1);ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin);DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin);RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39);ILPWKWPWWPWRR-NH2 (indolicidin); AAVALLPAVLLALLAP (RFGF); AALLPVLLAAP(RFGF analogue); and RKCRIVVIRVCR (bactenecin).

Exemplary cationic groups include, but are not limited to, protonatedamino groups, derived from e.g., O-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g.,O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino, ethylene diamine, polyamino); amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); and NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino).

As used herein the term “targeting ligand” refers to any molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. Some exemplary targeting ligandsinclude, but are not limited to, antibodies, antigens, folates, receptorligands, carbohydrates, aptamers, integrin receptor ligands, chemokinereceptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA,endothelin, GCPII, somatostatin, LDL and HDL ligands.

Carbohydrate based targeting ligands include, but are not limited to,D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc),multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalentmannose, multivalent lactose, N-acetyl-galactosamine,N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacidsand lectins. The term multivalent indicates that more than onemonosaccharide unit is present. Such monosaccharide subunits can belinked to each other through glycosidic linkages or linked to a scaffoldmolecule.

A number of folate and folate analogs amenable to the present inventionas ligands are described in U.S. Pat. Nos. 2,816,110; 51,410,104;5,552,545; 6,335,434 and 7,128,893, contents of which are hereinincorporated in their entireties by reference.

As used herein, the terms “PK modulating ligand” and “PK modulator”refers to molecules which can modulate the pharmacokinetics of thecomposition of the invention. Some exemplary PK modulator include, butare not limited to, lipophilic molecules, bile acids, sterols,phospholipid analogues, peptides, protein binding agents, vitamins,fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen,ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, andtransthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2, 4,6-triiodophenol and flufenamic acid). Oligomeric compounds that comprisea number of phosphorothioate intersugar linkages are also known to bindto serum protein, thus short oligomeric compounds, e.g. oligonucleotidesof comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides,preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality ofphosphorothioate linkages in the backbone are also amenable to thepresent invention as ligands (e.g. as PK modulating ligands). The PKmodulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more phosphorothioate and/orphosphorodithioate linkages. In some embodiments, all internucleotidelinkages in PK modulating oligonucleotide are phosphorothioate and/orphosphorodithioates linkages. In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also amenable to the presentinvention as PK modulating ligands. Binding to serum components (e.g.serum proteins) can be predicted from albumin binding assays, such asthose described in Oravcova, et al., Journal of Chromatography B (1996),677: 1-27.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

In some embodiments, ligand on one strand of a double-strandedoligomeric compound has affinity for a ligand on the second strand. Insome embodiments, a ligand is covalently linked to both strands of adouble-stranded oligomeric compound. As used herein, when a ligand islinked to more than oligomeric strand, point of attachment for anoligomeric compound can be an atom of the ligand self or an atom on acarrier molecule to which the ligand itself is attached.

Ligands can be coupled to the oligomeric compounds at various places,for example, 3′-end, 5′-end, and/or at an internal position. When two ormore ligands are present, the ligand can be on opposite ends of anoligomeric compound. In preferred embodiments, the ligand is attached tothe oligomeric compound via an intervening tether/linker. The ligand ortethered ligand can be present on a monomer when said monomer isincorporated into the growing strand. In some embodiments, the ligandcan be incorporated via coupling to a “precursor” monomer after said“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., monomer-linker-NH₂ can beincorporated into a growing oligomeric compound strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer into thestrand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor monomer by coupling the electrophilic group of theligand with the terminal nucleophilic group of the precursor monomer'stether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction can be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligomeric compounds, ligands can be attached to oneor both strands. In some embodiments, an siRNA comprises a ligandconjugated to the sense strand. In other embodiments, an siRNA comprisesa ligand conjugated to the antisense strand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of oligomeric compound.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. When a ligand is conjugated to anucleobase, the preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing.

Conjugation to sugar moieties of nucleosides can occur at any carbonatom. Example carbon atoms of a sugar moiety that can be attached to aconjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′position can also be attached to a conjugate moiety, such as in anabasic residue. Internucleosidic linkages can also bear conjugatemoieties. For phosphorus-containing linkages (e.g., phosphodiester,phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like),the conjugate moiety can be attached directly to the phosphorus atom orto an O, N, or S atom bound to the phosphorus atom. For amine- oramide-containing internucleosidic linkages (e.g., PNA), the conjugatemoiety can be attached to the nitrogen atom of the amine or amide or toan adjacent carbon atom.

Inventors have discovered inter alia that REVERSIR compounds conjugatedwith a ligand are particularly effective in reducing activity of siRNAs.Without wishing to be bound by a theory, a ligand can increase orenhance the ability of a REVERSIR compound by delivering the REVERSIRcompound to the desired location of action. Accordingly, in someembodiments, the REVERSIR compound is conjugated with a ligand.

While useful in delivery of the REVERSIR compound to a desired locationof action, the ligand conjugated with the REVERSIR compound cannegatively affect the ability of the REVERSIR compound to reduce siRNAactivity. Therefore, in some embodiments, the linkage between the ligandand the REVERSIR compound can be designed to undergo cleavage after theREVERSIR compound reaches a desired location of action. This can beaccomplished in a number of ways. For example, the linker connecting theREVERSIR compound to the ligand can be a cleavable linker.

The inventors have also discovered that the nucleoside in the REVERSIRcompound that is connected with the ligand can have an effect on theability of the REVERSIR compound to reduce activity of the siRNA.Inventors have discovered that ligand conjugated nucleosides comprisingdeoxy sugars (e.g., 2′-deoxy ribose) are particularly effective inenhancing the ability of REVERSIR compounds to reduce siRNA activity.Accordingly, in some embodiments, the nucleoside conjugated with theligand comprises a deoxy sugar, for example, a 2′-deoxy sugar.

In some embodiments of the various aspects disclosed herein, the ligandis attached to the nucleoside at the 3′-terminus of the REVERSIRcompound. The inventors have discovered inter alia that internucleotidelinkage between the ligand conjugated nucleotide and the rest of theREVERSIR compound can also have an effect on the ability of the REVERSIRcompound to reduce siRNA activity. Without wishing to be bound by atheory, readily cleavable internucleotide linkages were found to beparticularly effective in enhancing the ability of REVERSIR compounds toreduce siRNA activity. Accordingly, in some embodiments, the ligandconjugated nucleotide is attached to the rest of the REVERSIR compoundvia a cleavable internucleotide linage. In some embodiment, thecleavable internucleotide linkage is a phosphodiester internucleotidelinkage.

In some embodiments, the ligand conjugated nucleotide comprises a deoxysugar and is linked to rest of the REVERSIR compound via a cleavableinternucleotide linkage. In some further embodiments, of this thecleavable internucleotide linkage is a phosphodiester linkage.

In some embodiments, the ligand conjugated nucleotide comprises a deoxysugar and is linked to rest of the REVERSIR compound via aninternucleotide linkage that is not a phosphodiester linkage.

In some embodiments, the ligand is conjugated to the nucleotide at the3′-terminus of the REVERSIR compound.

In some embodiments, the ligand is conjugated at the 5′-terminus of theREVERSIR compound. In some embodiments, a first ligand is conjugated atthe 5′-terminus of the REVERSIR compound and a second ligand conjugatedto the first ligand.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of conjugates ofoligomeric compounds, e.g., oligonucleotides, include, but are notlimited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578, 717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578, 718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904, 582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082, 830; 5,112,963;5,149,782; 5,214,136; 5,245,022; 5,254, 469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510, 475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599, 923;5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153, 737; 6,172,208;6,300,319; 6,335,434; 6,335,437; 6,395, 437; 6,444,806; 6,486,308;6,525,031; 6,528,631; 6,559, 279; contents of which are hereinincorporated in their entireties by reference.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand having a structure shown below:

wherein:

-   -   L^(G) is independently for each occurrence a ligand, e.g.,        carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,        tetrasaccharide, polysaccharide; and    -   Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O        or S.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of Formula (II), (III), (IV) or (V):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q4^(A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;

Q and Q′ are independently for each occurrence is absent,—(P⁷-Q⁷-R⁷)_(p)-T⁷- or -T⁷-Q⁷-T^(7′)-B-T^(8′)-Q⁸-T⁸;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),P⁷, T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B),T^(5C), T⁷, T^(7′), T⁸ and T^(8′) are each independently for eachoccurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;B is —CH₂—N(B^(L))—CH₂—;B^(L) is -T^(B)-Q^(B)-T^(B′)-R^(x);

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C),Q⁷, Q⁸ and Q^(B) are independently for each occurrence absent, alkylene,substituted alkylene and wherein one or more methylenes can beinterrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)),C(R′)═C(R′), C≡C or C(O);

T^(B) and T^(B′) are each independently for each occurrence absent, CO,NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH₂, CH₂NH or CH₂O;

R^(x) is a lipophile (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid;

R¹, R², R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B),R^(5C), R⁷ are each independently for each occurrence absent, NH, O, S,CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L¹, L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) are each independently for each occurrence a carbohydrate, e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide and polysaccharide;

R′ and R″ are each independently H, C¹-C₆ alkyl, OH, SH, or N(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

R^(a) is H or amino acid side chain;

Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S; prepresent independently for each occurrence 0-20.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In some embodiments both L^(2A) and L^(2B) are different.

In some preferred embodiments both L^(3A) and L^(3B) are the same.

In some embodiments both L^(3A) and L^(3B) are different.

In some preferred embodiments both L^(4A) and L^(4B) are the same.

In some embodiments both L^(4A) and L^(4B) are different.

In some preferred embodiments all of L^(5A), L^(5B) and L^(5C) are thesame.

In some embodiments two of L^(5A), L^(5B) and L^(5C) are the same

In some embodiments L^(5A) and L^(5B) are the same.

In some embodiments L^(5A) and L^(5C) are the same.

In some embodiments L^(5B) and L^(5C) are the same.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein Y is O or S and n is 3-6.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein Y is O or S and n is 3-6.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein X is O or S.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer selected from the group consisting of:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is O or S.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

where in R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or NHCOOH.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

wherein R is OH or.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises amonomer of structure:

In the above described monomers, X and Y are each independently for eachoccurrence H, a protecting group, a phosphate group, a phosphodiestergroup, an activated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside,—P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, anucleotide, a nucleoside, or an oligonucleotide; and Z′ and Z″ are eachindependently for each occurrence O or S.

In certain embodiments, the REVERSIR compound is conjugated with aligand of structure:

In certain embodiments, the conjugated siRNA comprises a ligand ofstructure:

In certain embodiments, the REVERSIR compound comprises a monomer ofstructure:

Synthesis of above described ligands and monomers is described, forexample, in U.S. Pat. No. 8,106,022, content of which is incorporatedherein by reference in its entirety.

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand of structure:

In certain embodiments, the oligomeric compound described herein,including but not limited to REVERSIR compounds and siRNAs, comprises aligand from those described in U.S. Pat. No. 9,181,549 to Prakash etal., the content of which is incorporated herein by reference in itsentirety.

Linking groups or bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Linking groupsare useful for attachment of chemical functional groups, conjugategroups, reporter groups and other groups to selective sites in a parentcompound such as for example an oligomeric compound. In general abifunctional linking moiety comprises a hydrocarbyl moiety having twofunctional groups. One of the functional groups is selected to bind to aparent molecule or compound of interest and the other is selected tobind essentially any selected group such as chemical functional group ora conjugate group. In some embodiments, the linker comprises a chainstructure or an oligomer of repeating units such as ethylene glycol oramino acid units. Examples of functional groups that are routinely usedin a bifunctional linking moiety include, but are not limited to,electrophiles for reacting with nucleophilic groups and nucleophiles forreacting with electrophilic groups. In some embodiments, bifunctionallinking moieties include amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), and the like. Somenonlimiting examples of bifunctional linking moieties include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the ligand is conjugated with the oligomericcompound via a linker.

As used herein, the term “linker” means an organic moiety that connectstwo parts of a compound. Linkers typically comprise a direct bond or anatom such as oxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO,SO₂, SO₂NH or a chain of atoms, such as substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R¹)₂, C(O), cleavable linking group,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R¹ ishydrogen, acyl, aliphatic or substituted aliphatic.

In one embodiment, the linker is—[(P-Q″-R)_(q)—X—(P′—Q′″—R′)_(q′)]_(q″)-T-, wherein: P, R, T, P′, R′ andT are each independently for each occurrence absent, CO, NH, O, S,OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CH═N—O,

or heterocyclyl;Q″ and Q′″ are each independently for each occurrence absent,—(CH₂)_(n)—, —C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(n)C(R¹)(R²)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;X is absent or a cleavable linking group;R^(a) is H or an amino acid side chain;R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;n is independently for each occurrence 1-20; andm is independently for each occurrence 0-50.

In some embodiments, the linker comprises at least one cleavable linkinggroup.

In some embodiments, the linker is a branched linker. The branchpoint ofthe branched linker may be at least trivalent, but can be a tetravalent,pentavalent or hexavalent atom, or a group presenting such multiplevalencies. In some embodiments, the branchpoint is, —N, —N(Q)-C, —O—C,—S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or —N(Q)C(O)O—C;wherein Q is independently for each occurrence H or optionallysubstituted alkyl. In some embodiments, the branchpoint is glycerol orderivative thereof.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood or serum of a subject, orunder a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; amidases; endosomes or agents that cancreate an acidic environment, e.g., those that result in a pH of five orlower; enzymes that can hydrolyze or degrade an acid cleavable linkinggroup by acting as a general acid, peptidases (which can be substratespecific) and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In some embodiments, cleavable linking group is cleaved at least 1.25,1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in the cell (orunder in vitro conditions selected to mimic intracellular conditions) ascompared to blood or serum (or under in vitro conditions selected tomimic extracellular conditions). In some embodiments, the cleavablelinking group is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 5%, or 1% in the blood (or in vitro conditions selected tomimic extracellular conditions) as compared to in the cell (or under invitro conditions selected to mimic intracellular conditions).

Exemplary cleavable linking groups include, but are not limited to,redox cleavable linking groups (e.g., —S—S— and —C(R)₂—S—S—, wherein Ris H or C1-C6 alkyl and at least one R is C₁-C₆ alkyl such as CH₃ orCH₂CH₃); phosphate-based cleavable linking groups (e.g., —O—P(O)(OR)—O—,—O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—, —O—P(O)(OR)—S—,—S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—, —O—P(O)(R)—O—,—O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—, —S—P(O)(R)—S—,—O—P(S)(R)—S—, —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—,—S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—,—S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—,—S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, wherein R is optionallysubstituted linear or branched C₁-C₁₀ alkyl); acid celavable linkinggroups (e.g., hydrazones, esters, and esters of amino acids, —C═NN— and—OC(O)—); ester-based cleavable linking groups (e.g., —C(O)O—);peptide-based cleavable linking groups, (e.g., linking groups that arecleaved by enzymes such as peptidases and proteases in cells, e.g.,—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and RB are the R groups of thetwo adjacent amino acids). A peptide based cleavable linking groupcomprises two or more amino acids. In some embodiments, thepeptide-based cleavage linkage comprises the amino acid sequence that isthe substrate for a peptidase or a protease found in cells.

In some embodiments, an acid cleavable linking group is cleaveable in anacidic environment with a pH od about 6.5 or lower (e.g., about 6-, 5.5,5.0, or lower), or by agents such as enzymes that can act as a generalacid.

In some embodiments, the linker is an oligonucleotide linker including,but not limited to, (N)_(n); wherein N is independently a modified orunmodified nucleotide and n is 1-23. In some embodiments, n is 1-10,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, theoligonucleotide linker is selected from the group consisting of GNRA,(G)₄, (U)₄, and (dT)₄, wherein N is a modified or unmodified nucleotideand R is a modified or unmodified purine nucleotide. Some of thenucleotides in the linker can be involved in base-pair interactions withother nucleotides in the linker. It will be appreciated by one of skillin the art that any oligonucleotide chemical modifications or variationsdescribe herein can be used in the oligonucleotide linker. In certainembodiments, the linker is dA.

Motifs

The present invention also includes oligomeric compounds which arechimeric oligomeric compounds. “Chimeric” oligomeric compounds or“chimeras,” in the context of this invention, are oligomeric compoundswhich contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a modified or unmodified nucleotide inthe case of an oligonucleotide. Chimeric oligomeric compounds can bedescribed as having a particular motif. In some embodiments, the motifsinclude, but are not limited to, an alternating motif, a gapped motif, ahemimer motif, a uniformly fully modified motif and a positionallymodified motif. As used herein, the phrase “chemically distinct region”refers to an oligomeric region which is different from other regions byhaving a modification that is not present elsewhere in the oligomericcompound or by not having a modification that is present elsewhere inthe oligomeric compound. An oligomeric compound can comprise two or morechemically distinct regions. As used herein, a region that comprises nomodifications is also considered chemically distinct.

A chemically distinct region can be repeated within an oligomericcompound. Thus, a pattern of chemically distinct regions in anoligomeric compound can be realized such that a first chemicallydistinct region is followed by one or more second chemically distinctregions. This sequence of chemically distinct regions can be repeatedone or more times. Preferably, the sequence is repeated more than onetime. Both strands of a double-stranded oligomeric compound can comprisethese sequences. Each chemically distinct region can actually compriseas little as a single monomers, e.g., nucleotides. In some embodiments,each chemically distinct region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17 or 18 monomers, e.g., nucleotides.

In some embodiments, alternating nucleotides comprise the samemodification, e.g. all the odd number nucleotides in a strand have thesame modification and/or all the even number nucleotides in a strandhave the similar modification to the first strand. In some embodiments,all the odd number nucleotides in an oligomeric compound have the samemodification and all the even numbered nucleotides have a modificationthat is not present in the odd number nucleotides and vice versa.

When both strands of a double-stranded oligomeric compound comprise thealternating modification patterns, nucleotides of one strand can becomplementary in position to nucleotides of the second strand which aresimilarly modified. In an alternative embodiment, there is a phase shiftbetween the patterns of modifications of the first strand, respectively,relative to the pattern of similar modifications of the second strand.Preferably, the shift is such that the similarly modified nucleotides ofthe first strand and second strand are not in complementary position toeach other.

In some embodiments, the first strand has an alternating modificationpattern wherein alternating nucleotides comprise a 2′-modification,e.g., 2′-O-Methyl modification. In some embodiments, the first strandcomprises an alternating 2′-O-Methyl modification and the second strandcomprises an alternating 2′-fluoro modification. In other embodiments,both strands of a double-stranded oligonucleotide comprise alternating2′-O-methyl modifications.

When both strands of a double-stranded oligonucleotide comprisealternating 2′-O-methyl modifications, such 2′-modified nucleotides canbe in complementary position in the duplex region. Alternatively, such2′-modified nucleotides may not be in complementary positions in theduplex region.

In some embodiments, the oligonucleotide comprises two chemicallydistinct regions, wherein each region is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides in length.

In other embodiments, the oligomeric compound comprises three chemicallydistinct region. The middle region is about 5-15, (e.g., 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15) nucleotide in length and each flanking or wingregion is independently 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10)nucleotides in length. All three regions can have differentmodifications or the wing regions can be similarly modified to eachother. In some embodiments, the wing regions are of equal length, e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long.

As used herein the term “alternating motif” refers to an oligomericcompound comprising a contiguous sequence of linked monomer subunitswherein the monomer subunits have two different types of sugar groupsthat alternate for essentially the entire sequence of the oligomericcompound. Oligomeric compounds having an alternating motif can bedescribed by the formula: 5′-A(-L-B-L-A)n(-L-B)nn-3′ where A and B aremonomelic subunits that have different sugar groups, each L is aninternucleoside linking group, n is from about 4 to about 12 and nn is 0or 1. This permits alternating oligomeric compounds from about 9 toabout 26 monomer subunits in length. This length range is not meant tobe limiting as longer and shorter oligomeric compounds are also amenableto the present invention. In one embodiment, one of A and B is a2′-modified nucleoside as provided herein.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” refers to the modification of a nucleoside andincludes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “type region” refers to a portion of an oligomericcompound wherein the nucleosides and internucleoside linkages within theregion all comprise the same type of modifications; and the nucleosidesand/or the internucleoside linkages of any neighboring portions includeat least one different type of modification. As used herein the term“uniformly fully modified motif” refers to an oligonucleotide comprisinga contiguous sequence of linked monomer subunits that each have the sametype of sugar group. In one embodiment, the uniformly fully modifiedmotif includes a contiguous sequence of nucleosides of the invention. Inone embodiment, one or both of the 3′ and 5′-ends of the contiguoussequence of the nucleosides provided herein, comprise terminal groupssuch as one or more unmodified nucleosides.

As used herein the term “hemimer motif” refers to an oligomeric compoundhaving a short contiguous sequence of monomer subunits having one typeof sugar group located at the 5′ or the 3′ end wherein the remainder ofthe monomer subunits have a different type of sugar group. In general, ahemimer is an oligomeric compound of uniform sugar groups furthercomprising a short region (1, 2, 3, 4 or about 5 monomelic subunits)having uniform but different sugar groups and located on either the 3′or the 5′ end of the oligomeric compound. In one embodiment, the hemimermotif comprises a contiguous sequence of from about 10 to about 28monomer subunits of one type with from 1 to 5 or from 2 to about 5monomer subunits of a second type located at one of the termini. In oneembodiment, a hemimer is a contiguous sequence of from about 8 to about20 β-D-2′-deoxyribonucleosides having from 1-12 contiguous nucleosidesof the invention located at one of the termini. In one embodiment, ahemimer is a contiguous sequence of from about 8 to about 20β-D-2′-deoxyribonucleosides having from 1-5 contiguous nucleosides ofthe invention located at one of the termini. In one embodiment, ahemimer is a contiguous sequence of from about 12 to about 18β-D-2′-deoxyribo-nucleosides having from 1-3 contiguous nucleosides ofthe invention located at one of the termini. In one embodiment, ahemimer is a contiguous sequence of from about 10 to about 14β-D-2′-deoxyribonucleosides having from 1-3 contiguous nucleosides ofthe invention located at one of the termini.

As used herein the term “blockmer motif” refers to an oligonucleotidecomprising an otherwise contiguous sequence of monomer subunits whereinthe sugar groups of each monomer subunit is the same except for aninterrupting internal block of contiguous monomer subunits having adifferent type of sugar group. A blockmer overlaps somewhat with agapmer in the definition but typically only the monomer subunits in theblock have non-naturally occurring sugar groups in a blockmer and onlythe monomer subunits in the external regions have non-naturallyoccurring sugar groups in a gapmer with the remainder of monomersubunits in the blockmer or gapmer being β-D-2′-deoxyribonucleosides orβ-D-ribonucleosides. In one embodiment, blockmer oligonucleotides areprovided herein wherein all of the monomer subunits comprisenon-naturally occurring sugar groups.

As used herein the term “positionally modified motif” is meant toinclude an otherwise contiguous sequence of monomer subunits having onetype of sugar group that is interrupted with two or more regions of from1 to about 5 contiguous monomer subunits having another type of sugargroup. Each of the two or more regions of from 1 to about 5 contiguousmonomer subunits are independently uniformly modified with respect tothe type of sugar group. In one embodiment, each of the two or moreregions have the same type of sugar group. In one embodiment, each ofthe two or more regions have a different type of sugar group. In oneembodiment, positionally modified oligonucleotides are providedcomprising a sequence of from 8 to 20 β-D-2′-deoxyribonucleosides thatfurther includes two or three regions of from 2 to about 5 contiguousnucleosides of the invention. Positionally modified oligonucleotides aredistinguished from gapped motifs, hemimer motifs, blockmer motifs andalternating motifs because the pattern of regional substitution definedby any positional motif does not fit into the definition provided hereinfor one of these other motifs. The term positionally modified oligomericcompound includes many different specific substitution patterns.

As used herein the term “gapmer” or “gapped oligomeric compound” refersto an oligomeric compound having two external regions or wings and aninternal region or gap. The three regions form a contiguous sequence ofmonomer subunits with the sugar groups of the external regions beingdifferent than the sugar groups of the internal region and wherein thesugar group of each monomer subunit within a particular region is thesame. When the sugar groups of the external regions are the same thegapmer is a symmetric gapmer and when the sugar group used in the5′-external region is different from the sugar group used in the3′-external region, the gapmer is an asymmetric gapmer. In oneembodiment, the external regions are small (each independently 1, 2, 3,4 or about 5 monomer subunits) and the monomer subunits comprisenon-naturally occurring sugar groups with the internal region comprisingβ-D-2′-deoxyribonucleosides. In one embodiment, the external regionseach, independently, comprise from 1 to about 5 monomer subunits havingnon-naturally occurring sugar groups and the internal region comprisesfrom 6 to 18 unmodified nucleosides. The internal region or the gapgenerally comprises β-D-2′-deoxyribo-nucleosides but can comprisenon-naturally occurring sugar groups.

In one embodiment, the gapped oligomeric compounds comprise an internalregion of β-D-2′-deoxyribonucleosides with one of the external regionscomprising nucleosides of the invention. In one embodiment, the gappedoligonucleotide comprise an internal region ofβ-D-2′-deoxyribonucleosides with both of the external regions comprisingnucleosides of the invention. In one embodiment, the gappedoligonucleotide comprise an internal region ofβ-D-2′-deoxyribonucleosides with both of the external regions comprisingnucleosides of the invention. In one embodiment, gapped oligonucleotidesare provided herein wherein all of the monomer subunits comprisenon-naturally occurring sugar groups. In one embodiment, gappedoliogonucleotides are provided comprising one or two nucleosides of theinvention at the 5′-end, two or three nucleosides of the invention atthe 3′-end and an internal region of from 10 to 16β-D-2′-deoxyribonucleosides. In one embodiment, gapped oligonucleotidesare provided comprising one nucleoside of the invention at the 5′-end,two nucleosides of the invention at the 3′-end and an internal region offrom 10 to 16 β-D-2′-deoxyribonucleosides. In one embodiment, gappedoligonucleotides are provided comprising two nucleosides of theinvention at the 5′-end, two nucleosides of the invention at the 3′-endand an internal region of from 10 to 14 β-D-2′-deoxyribonucleosides. Inone embodiment, gapped oligonucleotides are provided that are from about10 to about 21 monomer subunits in length. In one embodiment, gappedoligonucleotides are provided that are from about 12 to about 16 monomersubunits in length. In one embodiment, gapped oligonucleotides areprovided that are from about 12 to about 14 monomer subunits in length.

In certain embodiments, the 5′-terminal monomer of an oligomericcompound of the invention comprises a phosphorous moiety at the 5′-end.In certain embodiments the 5′-terminal monomer comprises a2′-modification. In certain such embodiments, the 2′-modification of the5′-terminal monomer is a cationic modification. In certain embodiments,the 5′-terminal monomer comprises a 5′-modification. In certainembodiments, the 5′-terminal monomer comprises a 2′-modification and a5′-modification. In certain embodiments, the 5′-terminal monomer is a5′-stabilizing nucleoside. In certain embodiments, the modifications ofthe 5′-terminal monomer stabilize the 5′-phosphate. In certainembodiments, oligomeric compounds comprising modifications of the5′-terminal monomer are resistant to exonucleases. In certainembodiments, oligomeric compounds comprising modifications of the5′-terminal monomer have improved REVERSIR properties. In certain suchembodiments, oligomeric compound comprising modifications of the5′-terminal monomer have improved association with a strand of thesiRNA.

In certain embodiments, the 5′terminal monomer is attached to rest ofthe oligomeric compound a modified linkage. In certain such embodiments,the 5′terminal monomer is attached to rest of the oligomeric compound bya phosphorothioate linkage.

In certain embodiments, oligomeric compounds of the present inventioncomprise one or more regions of alternating modifications. In certainembodiments, oligomeric compounds comprise one or more regions ofalternating nucleoside modifications. In certain embodiments, oligomericcompounds comprise one or more regions of alternating linkagemodifications. In certain embodiments, oligomeric compounds comprise oneor more regions of alternating nucleoside and linkage modifications.

In certain embodiments, oligomeric compounds of the present inventioncomprise one or more regions of alternating 2′-F modified nucleosidesand 2′-OMe modified nucleosides. In certain such embodiments, suchregions of alternating 2′F modified and 2′OMe modified nucleosides alsocomprise alternating linkages. In certain such embodiments, the linkagesat the 3′ end of the 2′-F modified nucleosides are phosphorothioatelinkages. In certain such embodiments, the linkages at the 3′end of the2′OMe nucleosides are phosphodiester linkages.

In certain embodiments, such alternating regions are:

(2′-F)—(PS)-(2′-OMe)-(PO)

In certain embodiments, oligomeric compounds comprise 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 such alternating regions. Such regions may be contiguousor may be interrupted by differently modified nucleosides or linkages.

In certain embodiments, one or more alternating regions in analternating motif include more than a single nucleoside of a type. Forexample, oligomeric compounds of the present invention may include oneor more regions of any of the following nucleoside motifs:

ABA;

ABBA;

AABA;

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type. In certain embodiments, A and B are each selected from2′-F, 2′-OMe, LNA, DNA and MOE.

In certain embodiments, A is DNA. In certain embodiments B is DNA. Insome embodiments, A is 4′-CH₂O-2′-LNA. In certain embodiments, B is4′-CH₂O-2′-LNA. In certain embodiments, A is DNA and B is4′-CH₂O-2′-LNA. In certain embodiments A is 4′-CH₂O-2′-LNA and B is DNA.

In certain embodiments, A is 2′-OMe. In certain embodiments B is 2′-OMe.In certain embodiments, A is 2′-OMe and B is 4′-CH₂O-2′-LNA. In certainembodiments A is 4′-CH₂O-2′-LNA and B is 2′-OMe. In certain embodiments,A is 2′-OMe and B is DNA. In certain embodiments A is DNA and B is2′-OMe.

In certain embodiments, A is (S)-cEt. In some embodiments, B is (S)-cEt.In certain embodiments, A is 2′-OMe and B is (S)-cEt. In certainembodiments A is (S)-cEt and B is 2′-OMe. In certain embodiments, A isDNA and B is (S)-cEt. In certain embodiments A is (S)-cEt and B is DNA.

In certain embodiments, A is 2′-F. In certain embodiments B is 2′-F. Incertain embodiments, A is 2′-F and B is 4′-CH₂O-2′-LNA. In certainembodiments A is 4′-CH₂O-2′-LNA and B is 2′-F. In certain embodiments, Ais 2′-F and B is (S)-cEt. In certain embodiments A is (S)-cEt and B is2′-F. In certain embodiments, A is 2′-F and B is DNA. In certainembodiments A is DNA and B is 2′-F. In certain embodiments, A is 2′-OMeand B is 2′-F. In certain embodiments, A is DNA and B is 2′-OMe. Incertain embodiments, A is 2′-OMe and B is DNA.

In certain embodiments, oligomeric compounds having such an alternatingmotif also comprise a 5′ terminal nucleoside comprising a phosphatestabilizing modification. In certain embodiments, oligomeric compoundshaving such an alternating motif also comprise a 5′ terminal nucleosidecomprising a 2′-cationic modification. In certain embodiments,oligomeric compounds having such an alternating motif also comprise a 5′terminal modification.

Two-Two-Three Motifs

In certain embodiments, oligomeric compounds of the present inventioncomprise a region having a 2-2-3 motif. Such regions comprises thefollowing motif:

5′-(E)_(w)-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-(D)_(z)

wherein: A is a first type of modified nucleoside;

B, C, D, and E are nucleosides that are differently modified than A,however, B, C, D, and E may have the same or different modifications asone another;

w and z are from 0 to 15;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certainembodiments, B, C, D, and E are all 2′-F modified nucleosides. Incertain embodiments, A is a 2′-OMe modified nucleoside and B, C, D, andE are all 2′-F modified nucleosides.

In certain embodiments, the linkages of a 2-2-3 motif are all modifiedlinkages. In certain embodiments, the linkages are all phosphorothioatelinkages. In certain embodiments, the linkages at the 3′-end of eachmodification of the first type are phosphodiester.

In certain embodiments, Z is 0. In such embodiments, the region of threenucleosides of the first type are at the 3′-end of the oligonucleotide.In certain embodiments, such region is at the 3′-end of the oligomericcompound, with no additional groups attached to the 3′ end of the regionof three nucleosides of the first type. In certain embodiments, anoligomeric compound comprising an oligonucleotide where Z is 0, maycomprise a terminal group attached to the 3′-terminal nucleoside. Suchterminal groups may include additional nucleosides. Such additionalnucleosides are typically non-hybridizing nucleosides.

In certain embodiments, Z is 1-3. In certain embodiments, Z is 2. Incertain embodiments, the nucleosides of Z are 2′-MOE nucleosides. Incertain embodiments, Z represents non-hybridizing nucleosides. To avoidconfusion, it is noted that such non-hybridizing nucleosides might alsobe described as a 3′-terminal group with Z═O.

Combination Motifs

It is to be understood, that certain of the above described motifs andmodifications can be combined. Since a motif may comprises only a fewnucleosides, a particular oligomeric compound can comprise two or moremotifs. By way of non-limiting example, in certain embodiments,oligomeric compounds can have two or more nucleoside motifs selectedfrom LNAs, phosphorthioate linkages, 2′-OMe, conjugated ligand(s).

Oligomeric compounds having any of the various nucleoside motifsdescribed herein, can have also have any linkage motif. For example, inthe oligomeric compounds first 1, 2, 3, 4 or 5 at the 5′-end be modifiedintrersugar linkages and first 4, 5, 6, 7 or 8 intersugar linkages atthe 3′-end can be modified intersugar linkages. The central region ofsuch modified oligomeric compound can have intersugar linkages based onthe any of the other motifs described herein, for example, uniform,alternating, hemimer, gapmer, and the like. In some embodiments, theoligomeric compound comprise a phosphorothioate linkage between thefirst and second monomer at the 5′-terminus, alternatingphosphorothioate/phosphodiester linkages in the central region and 6, 7,or 8 phosphorothioate linkages at the 3′-terminus.

It is to be noted that the lengths of the regions defined by anucleoside motif and that of a linkage motif need not be the same.

In some embodiments, single-stranded oligomeric compounds or at leastone strand of a double-stranded oligomeric compound, includes at leastone of the following motifs:

-   -   (a) 5′-phosphorothioate or 5′-phosphorodithioate;    -   (b) a cationic modification of nucleotides 1 and 2 on the 5′        terminal, wherein the cationic modification is at C5 position of        pyrimidines and C2, C6, C8, exocyclic N2 or exocyclic N6 of        purines;    -   (c) at least one G-clamp nucleotide in the first two terminal        nucleotides at the 5′ end and the other nucleotide having a        cationic modification, wherein the cationic modification is at        C5 position of pyrimidines or C2, C6, C8, exocyclic N2 or        exocyclic N6 position of purines;    -   (d) at least one 2′-F modified nucleotide comprising a        nucleobase base modification;    -   (e) at least one gem-2′-O-methyl/2′-F modified nucleotide        comprising a nucleobase modification, preferably the methyl        substituent is in the up configuration, e.g. in the arabinose        configuration;    -   (f) a 5′-PuPu-3′ dinucleotide at the 3′ terminal wherein both        nucleotides comprise a modified MOE at 2′-position as described        in U.S. Patent Application Publication No. 20130130378, content        of which is incorporated herein by reference in its entirety.,    -   (g) a 5′-PuPu-3′ dinucleotide at the 5′ terminal wherein both        nucleotides comprise a modified MOE at 2′-position as described        in U.S. Patent Application Publication No. 20130130378;    -   (h) nucleotide at the 5′ terminal having a modified MOE at        2′-position as described in U.S. Patent Application Publication        No. 20130130378;    -   (i) nucleotide at the 5′ terminal having a 3′-F modification;    -   (j) 5′ terminal nucleotide comprising a 4′-substituent;    -   (k) 5′ terminal nucleotide comprising a 04′ modification;    -   (l) 3′ terminal nucleotide comprising a 4′-substituent; and    -   (m) combinations thereof.

In some embodiments, both strands of a double-stranded oligomericcompound independently comprise at least one of the above describedmotifs. In some other embodiments, both strands of a double-strandedoligomeric compound comprise at least one at least one of the abovedescribed motifs, which motifs can be same or different or somecombination of same and different.

The above examples are provided solely to illustrate how the describedmotifs may be used in combination and are not intended to limit theinvention to the particular combinations or the particular modificationsused in illustrating the combinations. Further, specific examplesherein, including, but not limited to those in the above table areintended to encompass more generic embodiments. For example, column A inthe above table exemplifies a region of alternating 2′-OMe and 2′-Fnucleosides. Thus, that same disclosure also exemplifies a region ofalternating different 2′-modifications. It also exemplifies a region ofalternating 2′-O-alkyl and 2′-halogen nucleosides. It also exemplifies aregion of alternating differently modified nucleosides. All of theexamples throughout this specification contemplate such genericinterpretation.

It is also noted that the lengths of oligomeric compounds, such as thoseexemplified in the above tables, can be easily manipulated bylengthening or shortening one or more of the described regions, withoutdisrupting the motif.

In some embodiments, oligomeric compound comprises two or morechemically distinct regions and has a structure as described inInternational Application No. PCT/US09/038433, filed Mar. 26, 2009,contents of which are herein incorporated in their entirety.

Synthesis, Purification and Analysis

Oligomerization of modified and unmodified nucleosides and nucleotidescan be routinely performed according to literature procedures for DNA(Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), HumanaPress) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA: Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57,5707-5713).

Oligomeric compounds provided herein can be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. It is well known to use similar techniques to prepareoligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of antisensecompound synthesis.

Methods of purification and analysis of oligomeric compounds are knownto those skilled in the art. Analysis methods include capillaryelectrophoresis (CE) and electrospray-mass spectroscopy. Such synthesisand analysis methods can be performed in multi-well plates. The methodof the invention is not limited by the method of oligomer purification.

The oligomeric compounds of the invention can be prepared usingsolution-phase or solid-phase organic synthesis, or enzymatically bymethods known in the art. Organic synthesis offers the advantage thatthe oligomeric strands comprising non-natural or modified nucleotidescan be easily prepared. Any other means for such synthesis known in theart can additionally or alternatively be employed. It is also known touse similar techniques to prepare other oligomeric compounds, such asthose comprising phosphorothioates, phosphorodithioates and alkylatedderivatives of intersugar linkages. The double-stranded oligomericcompounds of the invention can be prepared using a two-step procedure.First, the individual strands of the double-stranded molecule areprepared separately. Then, the component strands are annealed.

Regardless of the method of synthesis, the oligomeric compounds can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the oligonmeric preparationcan be precipitated and redissolved in pure double-distilled water, andlyophilized. The dried oligomeric compound can then be resuspended in asolution appropriate for the intended formulation process.

Teachings regarding the synthesis of particular modified oligomericcompounds can be found in the following U.S. patents or pending patentapplications: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone-modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof, U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N—2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingbeta-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups can be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.Nos. 5,223,168, and 5,608,046, both drawn to conjugated 4′-desmethylnucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn tobackbone-modified oligonucleotide analogs; and U.S. Pat. Nos. 6,262,241,and 5,459,255, drawn to, inter alia, methods of synthesizing2′-fluoro-oligonucleotides.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligomeric compounds can be admixed with pharmaceutically acceptableactive and/or inert substances for the preparation of pharmaceuticalcompositions or formulations. Compositions and methods for theformulation of pharmaceutical compositions are dependent upon a numberof criteria, including, but not limited to, route of administration,extent of disease, or dose to be administered.

Oligomeric compounds, including siRNAs and/or REVERSIR compounds, can beutilized in pharmaceutical compositions by combining such oligomericcompounds with a suitable pharmaceutically acceptable diluent orcarrier. A pharmaceutically acceptable diluent includesphosphate-buffered saline (PBS). PBS is a diluent suitable for use incompositions to be delivered parenterally. Accordingly, in oneembodiment, employed in the methods described herein is a pharmaceuticalcomposition comprising an antisense compound and/or antidote compoundand a pharmaceutically acceptable diluent. In certain embodiments, thepharmaceutically acceptable diluent is PBS.

Pharmaceutical compositions comprising oligomeric compounds encompassany pharmaceutically acceptable salts, esters, or salts of such esters.In certain embodiments, pharmaceutical compositions comprisingoligomeric compounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active oligomeric compound.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The oligomeric compounds can be delivered in a manner to target aparticular tissue, such as the liver (e.g., the hepatocytes of theliver).

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Liposome compositions can be prepared by a variety of methods that areknown in the art. See e.g., U.S. Pat. Nos. 4,235,871; 4,737,323;4,897,355 and 5,171,678; published International Applications WO96/14057 and WO 96/37194; Felgner, P. L. et al., Proc. Natl. Acad. Sci.,USA (1987) 8:7413-7417, Bangham, et al. M. Mol. Biol. (1965) 23:238,Olson, et al. Biochim. Biophys. Acta (1979) 557:9, Szoka, et al. Proc.Natl. Acad. Sci. (1978) 75: 4194, Mayhew, et al. Biochim. Biophys. Acta(1984) 775:169, Kim, et al. Biochim. Biophys. Acta (1983) 728:339, andFukunaga, et al. Endocrinol. (1984) 115:757.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Lipid Particles

In some embodiments, the REVERSIR can be fully encapsulated in a lipidformulation, e.g., a LNP, or other nucleic acid-lipid particle. TheREVERSIR encapsulated in the lipid formulation can be unconjugated orconjugated with a ligand (i.e., a conjugated REVERSIR).

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to REVERSIR ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol,or a mixture thereof. The cationic lipid can comprise from about 20 mol% to about 50 mol % or about 40 mol % of the total lipid present in theparticle.

In some embodiments, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-REVERSIR nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described inInternational application no. PCT/US2009/061897, published asWO/2010/048536, which is herein incorporated by reference.

In some embodiments, the lipid-REVERSIR particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 REVERSIR/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (C₁₂), aPEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), or aPEG-distearyloxypropyl (C₁₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

Additional exemplary lipid-REVERSIR formulations are described in Table1 below.

TABLE 1 Exemplary lipid REVERSIR formulations cationiclipid/non-cationic lipid/cholesterol/PEG-lipid conjugate FormulationIonizable/Cationic Lipid Lipid:REVERSIR ratio LNP_DLinDMA1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:REVERSIR~7:12-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:REVERSIR~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:REVERSIR~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:REVERSIR~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:REVERSIR~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:REVERSIR~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5Lipid:REVERSIR 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:REVERSIR10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:REVERSIR 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:REVERSIR10:1 yl)ethylazanediyl)didodecan-2-ol (C12-200> LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:REVERSIR: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:REVERSIR: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG- DSG 50/10/35/4.5/0.5 Lipid:REVERSIR:11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:REVERSIR: 7:1LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:REVERSIR: 10:1LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:REVERSIR: 12:1LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:REVERSIR: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:REVERSIR: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:REVERSIR: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:REVERSIR: 10:1 LNPX(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-(13Z,16Z)-N,N-dimethyl-3-nonyldocosa- 13,16-dien-1-amine13,16-dien-1-amine/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:REVERSIR: 10:1LNPY Biodegradable lipid Biodegradable lipid/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:REVERSIR: 10:1 *The REVERSIR can be an unconjugatedor conjugated with a ligand (i.e., conjugated REVERSIR).

Abbreviations in Table 1 include the following: DSPC:distearoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine;PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000); PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000); PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of2000).

DLinDMA (1,2-Dilinolenyloxy-N,N-dimethylaminopropane) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

Biodegradable lipid comprising formulations are described, e.g., PCTPublications No. WO2011/153493, filed Jun. 3, 2011 and WO/2013/086354,filed Dec. 7, 2012, the entire contents of which are hereby incorporatedby reference.

(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine comprisingformulations are described, e.g., in PCT Publications No.WO/2012/040184, filed Sep. 20, 2011, the entire contents of which arehereby incorporated by reference.

The oligomeric compounds of the invention can be prepared and formulatedas micelles. As used herein, “micelles” are a particular type ofmolecular assembly in which amphipathic molecules are arranged in aspherical structure such that all hydrophobic portions on the moleculesare directed inward, leaving the hydrophilic portions in contact withthe surrounding aqueous phase. The converse arrangement exists if theenvironment is hydrophobic.

In some embodiments, the formulations comprises micelles formed from anoligonucleotide of the invention and at least one amphiphilic carrier,in which the micelles have an average diameter of less than about 100nm, preferably. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

Micelle formulations can be prepared by mixing an aqueous solution ofthe oligonucleotide composition, an alkali metal C₈ to C₂₂ alkylsulphate, and an amphiphilic carrier. The amphiphilic carrier can beadded at the same time or after addition of the alkali metal alkylsulphate. Micelles will form with substantially any kind of mixing ofthe ingredients but vigorous mixing in order to provide smaller sizemicelles.

The oligomeric compounds of the present invention can be prepared andformulated as emulsions. As used herein, “emulsion” is a heterogenoussystem of one liquid dispersed in another in the form of droplets.

Emulsions are often biphasic systems comprising two immiscible liquidphases intimately mixed and dispersed with each other. In general,emulsions may be of either the water-in-oil (w/o) or the oil-in-water(o/w) variety. When an aqueous phase is finely divided into anddispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials is also included inemulsion formulations and contributes to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

In some embodiments, the compositions are formulated as microemulsions.As used herein, “microemulsion” refers to a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Microemuslions also include thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules.

A microemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature, for example see Idson, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245; and Block, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 335, contents of which are hereinincorporated by reference in their entirety.

The oligomeric compounds of the present invention can be prepared andformulated as lipid particles, e.g., formulated lipid particles (FLiPs)comprising (a) an oligonucleotide of the invention, where saidoligonucleotide has been conjugated to a lipophile and (b) at least onelipid component, for example an emulsion, liposome, isolatedlipoprotein, reconstituted lipoprotein or phospholipid, to which theconjugated oligonucleotide has been aggregated, admixed or associated.The stoichiometry of oligonucleotide to the lipid component can be 1:1.Alternatively the stoichiometry can be 1:many, many:1 or many:many,where many is two or more.

The FLiP can comprise triacylglycerols, phospholipids, glycerol and oneor several lipid-binding proteins aggregated, admixed or associated viaa lipophilic linker molecule with an oligonucleotide. Surprisingly, ithas been found that due to said one or several lipid-binding proteins incombination with the above mentioned lipids, the FLiPs show affinity toliver, gut, kidney, steroidogenic organs, heart, lung and/or muscletissue. These FLiPs can therefore serve as carrier for oligonucleotidesto these tissues. For example, lipid-conjugated oligonucleotides, e.g.,cholesterol-conjugated oligonucleotides, bind to HDL and LDL lipoproteinparticles which mediate cellular uptake upon binding to their respectivereceptors thus directing oligonucleotide delivery into liver, gut,kidney and steroidogenic organs, see Wolfrum et al. Nature Biotech.(2007), 25:1145-1157.

The FLiP can be a lipid particle comprising 15-25% triacylglycerol,about 0.5-2% phospholipids and 1-3% glycerol, and one or severallipid-binding proteins. FLiPs can be a lipid particle having about15-25% triacylglycerol, about 1-2% phospholipids, about 2-3% glycerol,and one or several lipid-binding proteins. In some embodiments, thelipid particle comprises about 20% triacylglycerol, about 1.2%phospholipids and about 2.25% glycerol, and one or several lipid-bindingproteins.

Another suitable lipid component for FLiPs is lipoproteins, for exampleisolated lipoproteins or more preferably reconstituted lipoproteins.Exemplary lipoproteins include chylomicrons, VLDL (Very Low DensityLipoproteins), IDL (Intermediate Density Lipoproteins), LDL (Low DensityLipoproteins) and HDL (High Density Lipoproteins). Methods of producingreconstituted lipoproteins are known in the art, for example see A.Jones, Experimental Lung Res. 6, 255-270 (1984), U.S. Pat. Nos.4,643,988 and 5,128,318, PCT publication WO87/02062, Canadian Pat. No.2,138,925. Other methods of producing reconstituted lipoproteins,especially for apolipoproteins A-I, A-II, A-IV, apoC and apoE have beendescribed in A. Jonas, Methods in Enzymology 128, 553-582 (1986) and G.Franceschini et al. J. Biol. Chem., 260(30), 16321-25 (1985).

One preferred lipid component for FLiP is Intralipid. Intralipid® is abrand name for the first safe fat emulsion for human use. Intralipid®20% (a 20% intravenous fat emulsion) is made up of 20% soybean oil, 1.2%egg yolk phospholipids, 2.25% glycerin, and water for injection. It isfurther within the present invention that other suitable oils, such assaflower oil, can serve to produce the lipid component of the FLiP.

FLiP can range in size from about 20-50 nm or about 30-50 nm, e.g.,about 35 nm or about 40 nm. In some embodiments, the FLiP has a particlesize of at least about 100 nm. FLiPs can alternatively be between about100-150 nm, e.g., about 110 nm, about 120 nm, about 130 nm, or about 140nm, whether characterized as liposome- or emulsion-based. Multiple FLiPscan also be aggregated and delivered together, therefore the size can belarger than 100 nm.

The process for making the lipid particles comprises the steps of: (a)mixing a lipid components with one or several lipophile (e.g.cholesterol) conjugated oligonucleotides that can be chemicallymodified; and (b) fractionating this mixture. In some embodiments, theprocess comprises the additional step of selecting the fraction withparticle size of 30-50 nm, preferably of about 40 nm in size.

Some exemplary lipid particle formulations amenable to the invention aredescribed in U.S. patent application Ser. No. 12/412,206, filed Mar. 26,2009, content of which is herein incorporated by reference in itsentirety.

In some embodiments, the oligomeric compounds can be formulated in yeastcell wall particles (“YCWP”). A yeast cell wall particle comprises anextracted yeast cell wall exterior and a core, the core comprising apayload (e.g., oligonucleotides). Exterior of the particle comprisesyeast glucans (e.g. beta glucans, beta-1,3-glucans, beta-1,6-glucans),yeast mannans, or combinations thereof. Yeast cell wall particles aretypically spherical particles about 1-4 μm in diameter.

Preparation of yeast cell wall particles is known in the art, and isdescribed, for example in U.S. Pat. Nos. 4,992,540; 5,082,936;5,028,703; 5,032,401; 5,322,841; 5,401,727; 5,504,079; 5,607,677;5,741,495; 5,830,463; 5,968,811; 6,444,448; and 6,476,003, U.S. Pat.App. Pub. Nos. 2003/0216346 and 2004/0014715, and Int. App. Pub. No. WO2002/12348, contents of which are herein incorporated by reference intheir entirety. Applications of yeast cell like particles for drugdelivery are described, for example in U.S. Pat. Nos. 5,032,401;5,607,677; 5,741,495; and 5,830,463, and U.S. Pat. Pub Nos. 2005/0281781and 2008/0044438, contents of which are herein incorporated by referencein their entirety. U.S. Pat. App. Pub. No. 2009/0226528, contents ofwhich are herein incorporated by reference, describes formulation ofnucleic acids with yeast cell wall particles for delivery ofoligonucleotide to cells.

Exemplary formulations for oligomeric compounds are described in U.S.Pat. Nos. 4,897,355; 4,394,448; 4,235,871; 4,231,877; 4,224,179;4,753,788; 4,673,567; 4,247,411; 4,814,270; 5,567,434; 5,552,157;5,565,213; 5,738,868; 5,795,587; 5,922,859; 6,077,663; 7,906,484; and8,642,076; PCT Publication No. WO2009/132131 and U.S. Pat. Pub. Nos.2006/0240093, 2007/0135372, 2011/0117125, 2009/0291131, 2012/0316220,2009/0163705 and 2013/0129785, contents of all of which is hereinincorporated by reference in its entirety. Behr (1994) BioconjugateChem. 5:382-389, and Lewis et al. (1996) PNAS 93:3176-3181), alsodescribe formulations for oligonucleotides that are amenable to theinvention, contents of which are herein incorporated by reference intheir entirety.

siRNA

As used herein, the term “siRNA” refers to an agent that mediates thetargeted cleavage of an RNA transcript. These agents associate with acytoplasmic multi-protein complex known as RNAi-induced silencingcomplex (RISC). Agents that are effective in inducing RNA interferenceare also referred to as siRNA, RNAi agent, or iRNA agent, herein. Asused herein, the term siRNA includes microRNAs and pre-microRNAs.

As used herein, the term “siRNA” refers to an agent that mediates thetargeted cleavage of an RNA transcript. These agents associate with acytoplasmic multi-protein complex known as RNAi-induced silencingcomplex (RISC). Agents that are effective in inducing RNA interferenceare also referred to as siRNA, dsRNA, RNAi agent, or iRNA agent herein.

As used herein, the terms “siRNA activity” and “RNAi activity” refer togene silencing by an siRNA.

As used herein, “gene silencing” by a RNA interference molecule refersto a decrease in the mRNA level in a cell for a target gene by at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, atleast about 99% up to and including 100%, and any integer in between ofthe mRNA level found in the cell without the presence of the miRNA orRNA interference molecule. In one preferred embodiment, the mRNA levelsare decreased by at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 99%, up to and including 100%and any integer in between 5% and 100%.”

As used herein the term “modulate gene expression” means that expressionof the gene, or level of RNA molecule or equivalent RNA moleculesencoding one or more proteins or protein subunits is up regulated ordown regulated, such that expression, level, or activity is greater thanor less than that observed in the absence of the modulator. For example,the term “modulate” can mean “inhibit,” but the use of the word“modulate” is not limited to this definition.

As used herein, gene expression modulation happens when the expressionof the gene, or level of RNA molecule or equivalent RNA moleculesencoding one or more proteins or protein subunits is at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold,5-fold or more different from that observed in the absence of the siRNA,e.g., RNAi agent. The % and/or fold difference can be calculatedrelative to the control or the non-control, for example,

${\%\mspace{14mu}{difference}} = \frac{\left\lbrack {{{expression}\mspace{14mu}{with}\mspace{14mu}{siRNA}} - {{expression}\mspace{14mu}{without}\mspace{14mu}{siRNA}}} \right\rbrack}{{expression}\mspace{14mu}{without}\mspace{14mu}{siRNA}}$  or${\%\mspace{14mu}{difference}} = \frac{\left\lbrack {{{expression}\mspace{14mu}{with}\mspace{14mu}{siRNA}} - {{expression}\mspace{14mu}{without}\mspace{14mu}{siRNA}}} \right\rbrack}{{expression}\mspace{14mu}{without}\mspace{14mu}{siRNA}}$

As used herein, the term “inhibit”, “down-regulate”, or “reduce” inrelation to gene expression, means that the expression of the gene, orlevel of RNA molecules or equivalent RNA molecules encoding one or moreproteins or protein subunits, or activity of one or more proteins orprotein subunits, is reduced below that observed in the absence ofmodulator. The gene expression is down-regulated when expression of thegene, or level of RNA molecules or equivalent RNA molecules encoding oneor more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is reduced at least 10% lower relative toa corresponding non-modulated control, and preferably at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably,100% (i.e., no gene expression).

As used herein, the term “increase” or “up-regulate” in relation to geneexpression, means that the expression of the gene, or level of RNAmolecules or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits, is increased above that observed in the absence of modulator.The gene expression is up-regulated when expression of the gene, orlevel of RNA molecules or equivalent RNA molecules encoding one or moreproteins or protein subunits, or activity of one or more proteins orprotein subunits, is increased at least 10% relative to a correspondingnon-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold,1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold ormore.

The term “increased” or “increase” as used herein generally means anincrease by a statically significant amount; for the avoidance of anydoubt, “increased” means an increase of at least 10% as compared to areference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

The term “reduced” or “reduce” as used herein generally means a decreaseby a statistically significant amount. However, for avoidance of doubt,“reduced” means a decrease by at least 10% as compared to a referencelevel, for example a decrease by at least about 20%, or at least about30%, or at least about 40%, or at least about 50%, or at least about60%, or at least about 70%, or at least about 80%, or at least about 90%or up to and including a 100% decrease (i.e. absent level as compared toa reference sample), or any decrease between 10-100% as compared to areference level.

The skilled person is well aware that double-stranded oligonucleotidescomprising a duplex structure of between 20 and 23, but specifically 21,base pairs have been hailed as particularly effective in inducing RNAinterference (Elbashir et al., EMBO 2001, 20:6877-6888). However, othershave found that shorter or longer double-stranded oligonucleotides canbe effective as well.

The double-stranded oligonucleotides comprise two oligonucleotidestrands that are sufficiently complementary to hybridize to form aduplex structure. Generally, the duplex structure is between 15 and 30,more generally between 18 and 25, yet more generally between 19 and 24,and most generally between 19 and 21 base pairs in length. In someembodiments, longer double-stranded oligonucleotides of between 25 and30 base pairs in length are preferred. In some embodiments, shorterdouble-stranded oligonucleotides of between 10 and 15 base pairs inlength are preferred. In another embodiment, the double-strandedoligonucleotide is at least 21 nucleotides long.

In some embodiments, the double-stranded oligonucleotide comprises asense strand and an antisense strand, wherein the antisense RNA strandhas a region of complementarity which is complementary to at least apart of a target sequence, and the duplex region is 14-30 nucleotides inlength. Similarly, the region of complementarity to the target sequenceis between 14 and 30, more generally between 18 and 25, yet moregenerally between 19 and 24, and most generally between 19 and 21nucleotides in length.

The phrase “antisense strand” as used herein, refers to an oligomericcompound that is substantially or 100% complementary to a targetsequence of interest. The phrase “antisense strand” includes theantisense region of both oligomeric compounds that are formed from twoseparate strands, as well as unimolecular oligomeric compounds that arecapable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

The phrase “sense strand” refers to an oligomeric compound that has thesame nucleoside sequence, in whole or in part, as a target sequence suchas a messenger RNA or a sequence of DNA. The terms “sense strand” and“passenger strand” are used interchangeably herein.

By “specifically hybridizable” and “complementary” is meant that anucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes. In reference to the nucleic molecules of the present invention,the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percentcomplementarity indicates the percentage of contiguous residues in anucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” or 100% complementarity means that all thecontiguous residues of a nucleic acid sequence will hydrogen bond withthe same number of contiguous residues in a second nucleic acidsequence. Less than perfect complementarity refers to the situation inwhich some, but not all, nucleoside units of two strands can hydrogenbond with each other. “Substantial complementarity” refers topolynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

The term “off-target” and the phrase “off-target effects” refer to anyinstance in which an siRNA against a given target causes an unintendedaffect by interacting either directly or indirectly with another mRNAsequence, a DNA sequence or a cellular protein or other moiety. Forexample, an “off-target effect” may occur when there is a simultaneousdegradation of other transcripts due to partial homology orcomplementarity between that other transcript and the sense and/orantisense strand of an siRNA.

In some embodiments, the double-stranded region of a double-strandedoligomeric compound is equal to or at least, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidepairs in length.

In some embodiments, the antisense strand of a double-strandedoligomeric compound is equal to or at least 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of a double-stranded oligomericcompound is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, one strand has at least one stretch of 1-5single-stranded nucleotides in the double-stranded region. By “stretchof single-stranded nucleotides in the double-stranded region” is meantthat there is present at least one nucleotide base pair at both ends ofthe single-stranded stretch. In some embodiments, both strands have atleast one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-strandednucleotides in the double stranded region. When both strands have astretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides inthe double stranded region, such single-stranded nucleotides can beopposite to each other (e.g., a stretch of mismatches) or they can belocated such that the second strand has no single-stranded nucleotidesopposite to the single-stranded oligonucleotides of the first strand andvice versa (e.g., a single-stranded loop). In some embodiments, thesingle-stranded nucleotides are present within 8 nucleotides from eitherend, for example 8, 7, 6, 5, 4, 3, or 2 nucleotide from either the 5′ or3′ end of the region of complementarity between the two strands.

In some embodiments, each strand of the double-stranded oligonucleotidehas a ZXY structure, such as is described in PCT Publication No.2004080406, content of which is hereby incorporated in its entireties.

In certain embodiment, the two strands of double-stranded oligomericcompound can be linked together. The two strands can be linked to eachother at both ends, or at one end only. By linking at one end is meantthat 5′-end of first strand is linked to the 3′-end of the second strandor 3′-end of first strand is linked to 5′-end of the second strand. Whenthe two strands are linked to each other at both ends, 5′-end of firststrand is linked to 3′-end of second strand and 3′-end of first strandis linked to 5′-end of second strand. The two strands can be linkedtogether by an oligonucleotide linker including, but not limited to,(N)_(n); wherein N is independently a modified or unmodified nucleotideand n is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8,9, or 10. In some embodiments, the oligonucleotide linker is selectedfrom the group consisting of GNRA, (G)₄, (U)₄, and (dT)₄, wherein N is amodified or unmodified nucleotide and R is a modified or unmodifiedpurine nucleotide. Some of the nucleotides in the linker can be involvedin base-pair interactions with other nucleotides in the linker. The twostrands can also be linked together by a non-nucleosidic linker, e.g. alinker described herein. It will be appreciated by one of skill in theart that any oligonucleotide chemical modifications or variationsdescribe herein can be used in the oligonucleotide linker.

Hairpin and dumbbell type oligomeric compounds will have a duplex regionequal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or25 nucleotide pairs. The duplex region can be equal to or less than 200,100, or 50, in length. In some embodiments, ranges for the duplex regionare 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

The hairpin oligomeric compounds can have a single strand overhang orterminal unpaired region, in some embodiments at the 3′, and in someembodiments on the antisense side of the hairpin. In some embodiments,the overhangs are 1-4, more generally 2-3 nucleotides in length. Thehairpin oligomeric compounds that can induce RNA interference are alsoreferred to as “shRNA” herein.

In certain embodiments, two oligomeric strands specifically hybridizewhen there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringentconditions” refers to conditions under which an antisense compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, and “stringent conditions” underwhich antisense compounds hybridize to a target sequence are determinedby the nature and composition of the antisense compounds and the assaysin which they are being investigated.

It is understood in the art that incorporation of nucleotide affinitymodifications may allow for a greater number of mismatches compared toan unmodified compound. Similarly, certain oligonucleotide sequences maybe more tolerant to mismatches than other oligonucleotide sequences. Oneof ordinary skill in the art is capable of determining an appropriatenumber of mismatches between oligonucleotides, or between anoligonucleotide and a target nucleic acid, such as by determiningmelting temperature (Tm). Tm or ΔTm can be calculated by techniques thatare familiar to one of ordinary skill in the art. For example,techniques described in Freier et al. (Nucleic Acids Research, 1997, 25,22: 4429-4443) allow one of ordinary skill in the art to evaluatenucleotide modifications for their ability to increase the meltingtemperature of an RNA:DNA duplex.

Modulation of Target Expression

In certain embodiments, a target nucleic acid is a mRNA. In certain suchembodiments, siRNAs are designed to modulate that target mRNA or itsexpression. In certain embodiments, designing an antisense compound to atarget nucleic acid molecule can be a multistep process. Typically theprocess begins with the identification of a target protein, the activityof which is to be modulated, and then identifying the nucleic acid theexpression of which yields the target protein. In certain embodiments,designing of an antisense compound results in an antisense compound thatis hybridizable to the targeted nucleic acid molecule. In certainembodiments, the antisense compound is an antisense oligonucleotide orantisense oligonucleoside. In certain embodiments, an antisense compoundand a target nucleic acid are complementary to one another. In certainsuch embodiments, an antisense compound is perfectly complementary to atarget nucleic acid. In certain embodiments, an antisense compoundincludes one mismatch. In certain embodiments, an antisense compoundincludes two mismatches. In certain embodiments, an antisense compoundincludes three or more mismatches.

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid functions. In certainembodiments, the functions of RNA to be modulated include, but are notlimited to, translocation functions, which include, but are not limitedto, translocation of the RNA to a site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, and translation of protein from the RNA. RNAprocessing functions that can be modulated include, but are not limitedto, splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.Thus, in one embodiment modulation of expression can mean increase ordecrease in target RNA or protein levels. In another embodimentmodulation of expression can mean an increase or decrease of one or moreRNA splice products, or a change in the ratio of two or more spliceproducts.

In certain embodiments, the siRNA is a conjugated siRNA. As used herein,the term “conjugated siRNA” refers to an RNAi agent that is conjugatedwith a ligand. For Example, an RNAi agent conjugated with a liganddescribed herein.

In some other embodiments, the siRNA is an unconjugated siRNA. As usedherein, the term “unconjugated siRNA” refers to an RNAi agent that isnot conjugated with a ligand, e.g., a ligand described herein.

In one aspect, the invention relates to a double-stranded RNA (dsRNA)agent, i.e., siRNA, for inhibiting the expression of a target gene. ThedsRNA agent comprises a sense strand and an antisense strand, eachstrand having 14 to 40 nucleotides. The dsRNA agent is represented byformula (I):

In formula (I), B1, B2, B3, B1′, B2′, B3′, and B4′ each areindependently a nucleotide containing a modification selected from thegroup consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substitutedalkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′,B2′, B3′, and B4′ each contain 2′-OMe modifications.

C1 is a thermally destabilizing nucleotide placed at a site opposite tothe seed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). For example, C1 is at a position of thesense strand that pairs with a nucleotide at positions 2-8 of the 5′-endof the antisense strand. C1 nucleotide bears the thermally destabilizingmodification which can include abasic modification; mismatch with theopposing nucleotide in the duplex; and sugar modification such as2′-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids(UNA) or glycerol nuceltic acid (GNA). In one embodiment, C1 hasthermally destabilizing modification selected from the group consistingof: i) mismatch with the opposing nucleotide in the antisense strand;ii) abasic modification selected from the group consisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, thethermally destabilizing modification in C1 is a mismatch selected fromthe group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T,U:U, T:T, and U:T; and optionally, at least one nucleobase in themismatch pair is a 2′-deoxy nucleobase. In one example, the thermallydestabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a modification providing the nucleotide a steric bulk that isless or equal to the steric bulk of a 2′-OMe modification. Themodification can be at the 2′ position of a ribose sugar of thenucleotide, or a modification to a non-ribose nucleotide, acyclicnucleotide, or the backbone of the nucleotide that is similar orequivalent to the 2′ position of the ribose sugar, and provides thenucleotide a steric bulk that is less than or equal to the steric bulkof a 2′-OMe modification. For example, T1, T1′, T2′, and T3′ are eachindependently selected from DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl. Inone embodiment, T1 is DNA. In one embodiment, T1′ is DNA, RNA or LNA. Inone embodiment, T2′ is DNA or RNA. In one embodiment, T3′ is DNA or RNA.

n¹, n³, and q¹ are independently 4 to 15 nucleotides in length.

n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length.

n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length.

q⁵ is independently 0-10 nucleotide(s) in length.

n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with twophosphorothioate internucleotide linkage modifications within position1-5 of the sense strand (counting from the 5′-end of the sense strand),and two phosphorothioate internucleotide linkage modifications atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴, q², q⁴, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n⁴ is1.

In one embodiment, T3′ starts at position 2 from the 5′ end of theantisense strand. In one example, T3′ is at position 2 from the 5′ endof the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length(i.e. not counting the T1′ and T3′ nucleotides.

In one embodiment, T1′ is at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1, and the modification atthe 2′ position or positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisensestrand. In one example, T3′ is at position 2 from the 5′ end of theantisense strand and q⁶ is equal to 1, and the modification at the 2′position or positions in a non-ribose, acyclic or backbone that provideless than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at cleavage site of the sense strand. In oneexample, T1 is at position 11 from the 5′ end of the sense strand, whenthe sense strand is 19-22 nucleotides in length, and n² is 1.

In one embodiment, T2′ starts at position 6 from the 5′ end of theantisense strand. In one example, T2′ is at positions 6-10 from the 5′end of the antisense strand, and q⁴ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35% or 30% of the dsRNA agent of the invention ismodified.

In one embodiment, each of the sense and antisense strands of the dsRNAagent is independently modified with acyclic nucleotides, LNA, HNA,CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP),or 2′-ara-F.

In one embodiment, each of the sense and antisense strands of the dsRNAagent contains at least two different modifications.

In one embodiment, the dsRNA agent of Formula (I) further comprises 3′and/or 5′ overhang(s) of 1-10 nucleotides in length. In one example,dsRNA agent of formula (I) comprises a 3′ overhang at the 3′-end of theantisense strand and a blunt end at the 5′-end of the antisense strand.In another example, the dsRNA agent has a 5′ overhang at the 5′-end ofthe sense strand.

In one embodiment, the dsRNA agent of the invention does not contain any2′-F modification.

In one embodiment, the sense strand and/or antisense strand of the dsRNAagent comprises one or more blocks of phosphorothioate ormethylphosphonate internucleotide linkages. In one example, the sensestrand comprises one block of two phosphorothioate or methylphosphonateinternucleotide linkages. In one example, the antisense strand comprisestwo blocks of two phosphorothioate or methylphosphonate internucleotidelinkages. For example, the two blocks of phosphorothioate ormethylphosphonate internucleotide linkages are separated by 16-18phosphate internucleotide linkages.

In one embodiment, each of the sense and antisense strands of the dsRNAagent has 15-30 nucleotides. In one example, the sense strand has 19-22nucleotides, and the antisense strand has 19-25 nucleotides. In anotherexample, the sense strand has 21 nucleotides, and the antisense strandhas 23 nucleotides.

In one embodiment, the nucleotide at position 1 of the 5′-end of theantisense strand in the duplex is selected from the group consisting ofA, dA, dU, U, and dT. In one embodiment, at least one of the first,second, and third base pair from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the antisense strand of the dsRNA agent of theinvention is 100% complementary to a target RNA to hybridize thereto andinhibits its expression through RNA interference. In another embodiment,the antisense strand of the dsRNA agent of the invention is at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, or at least 50%complementary to a target RNA.

In one aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 40nucleotides. The sense strand contains at least one thermallydestabilizing nucleotide, wherein at at least one said thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand when the sense strand is 21 nucleotides in length. Theantisense strand contains at least two modified nucleic acids that aresmaller than a sterically demanding 2′-OMe modification. Preferably, thetwo modified nucleic acids that is smaller than a sterically demanding2′-OMe are separated by 11 nucleotides in length. For example, the twomodified nucleic acids are at positions 2 and 14 of the 5′end of theantisense strand.

In one embodiment, the sense strand sequence of the dsRNA agent isrepresented by formula (Is):

wherein:

-   -   B1, B2, and B3 each independently represent a nucleotide        containing a modification selected from the group consisting of        2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo,        ENA, and BNA/LNA;    -   C1 is a thermally destabilizing nucleotide (e.g., acyclic        nucleotide such as UNA or GNA, mismatch, abasic, or DNA) placed        at the opposite of the antisense seed region (i.e., positions        2-8 of the 5′-end of the antisense strand);    -   T1 represents a nucleotide comprising a chemical modification at        the 2′ position or equivalent position in a non-ribose, acyclic        or backbone that provide the nucleotide a less steric bulk than        a 2′-OMe modification; for example, T1 is selected from the        group consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹ or n³ is independently 4 to 15 nucleotides in length;    -   n⁵ is 1-6 nucleotide(s) in length;    -   n⁴ is 1-3 nucleotide(s) in length; and    -   n² is 0-3 nucleotide(s) in length.

In one embodiment, the sense strand sequence having 19, 20, 21, or 22nucleotides in length of the dsRNA agent is represented by formula (Is):

wherein:

-   -   B1, B2, and B3 each independently represent a nucleotide        containing a modification selected from the group consisting of        2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo,        ENA, and BNA/LNA;    -   C1 is a thermally destabilizing nucleotide (e.g., acyclic        nucleotide such as UNA or GNA, mismatch, abasic, or DNA) placed        at the opposite of the antisense seed region (i.e., positions        2-8 of the 5′-end of the antisense strand);    -   T1 represents a nucleotide comprising a chemical modification        selected from the group consisting of DNA, RNA, LNA, 2′-F, and        2′-F-5′-methyl;    -   n¹ or n³ is independently 4 to 15 nucleotides in length;    -   n⁵ is 1-6 nucleotide(s) in length;    -   n⁴ is 1-3 nucleotide(s) in length; and    -   n² is 0-3 nucleotide(s) in length.

In one embodiment, the dsRNA agent of formula (Is) further comprises 3′and/or 5′ overhang(s) of 1-10 nucleotides in length. In one example, thedsRNA agent of formula (Is) comprises a 5′ overhang.

In one embodiment, C1 comprises one thermally destabilizing nucleotideat position 14, 15, 16 or 17 from the 5′-end of the sense strand. Forexample, C1 is an acyclic nucleotide (e.g., UNA or GNA), mismatch,abasic, or DNA. In one specific example, C1 is a GNA.

In one embodiment, T1 comprises a DNA, RNA, LNA, 2′-F, or 2′-F-5′-methylat position 11 from the 5′-end of the sense strand.

In one embodiment, the dsRNA agent of the invention comprises a sensestrand (Is), wherein C1 is an acyclic nucleotide (e.g., UNA or GNA),mismatch, abasic, or DNA; and T1 comprises a DNA, RNA, LNA, 2′-F, or2′-F-5′-methyl at position 11 from the 5′-end of the sense strand.

In one embodiment, the antisense strand sequence of the dsRNA agent isrepresented by formula (Ia):

wherein:

-   -   B1′, B2′, B3′, and B4′ each independently represent a nucleotide        containing a modification selected from the group consisting of        2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo,        ENA, and BNA/LNA;    -   T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification at the 2′ position or        equivalent position in a non-ribose, acyclic or backbone that        provide the nucleotide a less steric bulk than a 2′-OMe        modification;    -   for example, T1′, T2′, and T3′ each are independently selected        from the group consisting of DNA, RNA, LNA, 2′-F, and        2′-F-5′-methyl;    -   q is independently 4 to 15 nucleotides in length;    -   q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   q⁴ is independently 0-3 nucleotide(s) in length; and    -   q⁵ is independently 0-10 nucleotide(s) in length.

In one embodiment, the antisense strand sequence having 19, 20, 21, 22,23, 24, or 25 nucleotides in length of the dsRNA agent is represented byformula (Ia):

wherein:

-   -   B1′, B2′, B3′, and B4′ each independently represent a nucleotide        containing a modification selected from the group consisting of        2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo,        ENA, and BNA/LNA;    -   T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   q is independently 4 to 15 nucleotides in length;    -   q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   q⁴ is independently 0-3 nucleotide(s) in length; and    -   q⁵ is independently 0-10 nucleotide(s) in length.

In one embodiment, dsRNA of formula (Ia) further comprises 3′ and/or 5′overhang(s) of 1-10 nucleotides in length. In one example, dsRNA offormula (Ia) comprises a 3′ overhang.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

-   -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification selected from the group        consisting of 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted        alkyl, 2′-halo, ENA, and BNA/LNA; C1 is an acyclic nucleotide        (e.g., UNA or GNA);    -   T1, T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has 3′ and/or 5′ overhang(s) of 1-10        nucleotides in length of the antisense and/or sense strand(s).

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

-   -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification selected from the group        consisting of 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted        alkyl, 2′-halo, ENA, and BNA/LNA; C1 is an acyclic nucleotide        (e.g., UNA or GNA);    -   T1, T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 3′ overhang of 2 nucleotides in        length at the 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 15-30 nucleotides:

wherein:

-   -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification 2′-OMe;    -   C1 is an acyclic nucleotide GNA;    -   T1, T1′, T2′, and T3′ each are independently DNA or RNA;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 3′ overhang of 1-6 nucleotides in        length at the 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 19-23 nucleotides:

wherein:

-   -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a 2′-OMe modification;    -   C1 is an acyclic nucleotide GNA;    -   T1, T1′, T2′, and T3′ are independently DNA or RNA;    -   n¹, n³, q¹, or q³ is independently 4 to 15 nucleotides in        length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n², q⁴ or q⁵ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 3′ overhang of 2 nucleotides in        length at the 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

-   -   wherein:    -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification selected from the group        consisting of 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted        alkyl, 2′-halo, ENA, and BNA/LNA;    -   C1 is an acyclic nucleotide (e.g., UNA or GNA);    -   T1, T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 5′ overhang of 1-10 nucleotides in        length at the 5′-end of the sense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

-   -   wherein:    -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification selected from the group        consisting of 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted        alkyl, 2′-halo, ENA, and BNA/LNA;    -   C1 is an acyclic nucleotide (e.g., UNA or GNA);    -   T1, T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 5′ overhang of 1-6 nucleotides in        length at the 5′-end of the sense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

-   -   wherein:    -   B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent        a nucleotide containing a modification selected from the group        consisting of 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted        alkyl, 2′-halo, ENA, and BNA/LNA;    -   C1 is an acyclic nucleotide (e.g., UNA or GNA);    -   T1, T1′, T2′, and T3′ each independently represent a nucleotide        comprising a chemical modification selected from the group        consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;    -   n¹, n³, or q¹ is independently 4 to 15 nucleotides in length;    -   n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;    -   n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length;    -   n² or q⁴ is independently 0-3 nucleotide(s) in length;    -   q⁵ is independently 0-10 nucleotide(s) in length; and    -   wherein the dsRNA agent has a 5′ overhang of 1-10 nucleotides in        length at the 5′-end of the sense and a 3′ overhang of 1-10        nucleotides in length at the 5′-end of the antisense strand.

Thermally Destabilizing Modifications

The dsRNA agent can be optimized for RNA interference by increasing thepropensity of the dsRNA duplex to disassociate or melt (decreasing thefree energy of duplex association) by introducing a thermallydestabilizing modification in the sense strand at a site opposite to theseed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). This modification can increase thepropensity of the duplex to disassociate or melt in the seed region ofthe antisense strand.

The thermally destabilizing modifications can include abasicmodification; mismatch with the opposing nucleotide in the opposingstrand; and sugar modification such as 2′-deoxy modification or acyclicnucleotide, e.g., unlocked nucleic acids (UNA) or glycerol nuceltic acid(GNA).

Exemplified abasic modifications are:

Exemplified sugar modifications are:

The term “acyclic nucleotide” refers to any nucleotide having an acyclicribose sugar, for example, where any of bonds between the ribose carbons(e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent and/orat least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′ orO4′) are independently or in combination absent from the nucleotide. Insome embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refersto unlocked acyclic nucleic acid, wherein any of the bonds of the sugarhas been removed, forming an unlocked “sugar” residue. In one example,UNA also encompasses monomers with bonds between C1′-C4′ being removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar isremoved (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059(1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which arehereby incorporated by reference in their entirety). The acyclicderivative provides greater backbone flexibility without affecting theWatson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similarto DNA or RNA but differing in the composition of its “backbone” in thatis composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification can be mismatches (i.e.,noncomplementary base pairs) between the thermally destabilizingnucleotide and the opposing nucleotide in the opposite strand within thedsRNA duplex. Exemplary mismatch basepairs include G:G, G:A, G:U, G:T,A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Othermismatch base pairings known in the art are also amenable to the presentinvention. A mismatch can occur between nucleotides that are eithernaturally occurring nucleotides or modified nucleotides, i.e., themismatch base pairing can occur between the nucleobases from respectivenucleotides independent of the modifications on the ribose sugars of thenucleotides. In certain embodiments, the dsRNA agent contains at leastone nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase;e.g., the 2′-deoxy nucleobase is in the sense strand.

More examples of abasic nucleotide, acyclic nucleotide modifications(including UNA and GNA), and mismatch modifications have been describedin detail in WO 2011/133876, which is herein incorporated by referencein its entirety.

The thermally destabilizing modifications may also include universalbase with reduced or abolished capability to form hydrogen bonds withthe opposing bases, and phosphate modifications.

Nucleobase modifications with impaired or completely abolishedcapability to form hydrogen bonds with bases in the opposite strand havebeen evaluated for destabilization of the central region of the dsRNAduplex as described in WO 2010/0011895, which is herein incorporated byreference in its entirety. Exemplary nucleobase modifications are:

Exemplary phosphate modifications known to decrease the thermalstability of dsRNA duplexes compared to natural phosphodiester linkagesare:

In one embodiment, the dsRNA agent of the invention can comprise 2′-5′linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S). For example,the 2′-5′ linkages modifications can be used to promote nucleaseresistance or to inhibit binding of the sense to the antisense strand,or can be used at the 5′ end of the sense strand to avoid sense strandactivation by RISC.

In another embodiment, the dsRNA agent of the invention can comprise Lsugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). Forexample, these L sugars modifications can be used to promote nucleaseresistance or to inhibit binding of the sense to the antisense strand,or can be used at the 5′ end of the sense strand to avoid sense strandactivation by RISC.

In one embodiment, the dsRNA agent is a multimer containing at least twoduplexes represented by formula (I), wherein said duplexes are connectedby a linker. The linker can be cleavable or non-cleavable. Optionally,said multimer further comprise a ligand. Each of the dsRNA agent cantarget the same gene or two different genes; or each of the dsRNA agentcan target same gene at two different target sites.

In one embodiment, the dsRNA agent is a multimer containing three, four,five, six or more duplexes represented by formula (I), wherein saidduplexes are connected by a linker. The linker can be cleavable ornon-cleavable. Optionally, said multimer further comprises a ligand.Each of the dsRNA agent can target the same gene or two different genes;or each of the dsRNA agent can target same gene at two different targetsites.

In one embodiment, two dsRNA agent represented by formula (I) are linkedto each other at the 5′ end, and one or both of the 3′ ends of the areoptionally conjugated to a ligand. Each of the dsRNA can target the samegene or two different genes; or each of the dsRNA can target same geneat two different target sites.

Various publications described multimeric siRNA and can all be used withthe dsRNA of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydratemoieties to a dsRNA agent can optimize one or more properties of thedsRNA agent. In many cases, the carbohydrate moiety will be attached toa modified subunit of the dsRNA agent. E.g., the ribose sugar of one ormore ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In one embodiment the dsRNA agent of the invention is conjugated to aligand via a carrier, wherein the carrier can be cyclic group or acyclicgroup; preferably, the cyclic group is selected from pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and decalin; preferably, the acyclic group is selectedfrom serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally beconjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand, in particular, the 3′-end of the sense strand.

In one embodiment dsRNA agents of the invention are 5′ phosphorylated orinclude a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S—5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substitutedvinyl), (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). In one example, the modification can in placed in theantisense strand of a dsRNA agent.

REVERSIR Compounds

In certain instances it is desirable to inhibit siRNA activity. Forexample, in certain embodiments where the siRNA target is an mRNA, it ismay be desirable to inhibit siRNA activity and thereby restoreexpression of a target protein. For example, certain siRNAs have beenused therapeutically. In certain such uses, siRNAs are long-acting. Incertain instances, such long acting siRNAs are desirable, for theirconvenience. In such instances, though, it can also be desirable to havea means to reverse the activity of an siRNA. For example, a patient mayrespond poorly to treatment or receive too high a dose. In such aninstance, a reverser compound can be administered to at least partiallyreduce the RNAi activity of the siRNA. In certain embodiments, thelong-lasting effect of siRNA makes waiting for that effect to slowlydiminish through natural clearance an unattractive option.

By way of example, and without limiting the present invention, certainsiRNAs are useful for inhibiting blood clotting factors (e.g., Factor II(prothrombin), Factor VII, Factor IX, etc.). Such siRNAs havetherapeutic potential as anticoagulants. Long half-lives make suchsiRNAs particularly attractive, however, if a patient receives too higha dose, has surgery (where anti-coagulation is undesirable) or otherwisedesires a decrease in the anti-coagulant effect, a reverser compound tothe anti-coagulant siRNA can be administered. Such REVERSIR compoundwill restore coagulation function more quickly than simply waiting fornatural clearance of the siRNA. This example is provided forillustrative purposes. Many siRNAs have been designed to a vast numberof targets, including without limitation, a vast number of messenger RNA(mRNA) targets and pre-mRNA targets, as well as a vast number ofnon-coding RNA targets. REVERSIR compounds provided herein are suitablefor any siRNA, regardless of the target or mechanism of the siRNAcompound.

In certain embodiments, the invention provides REVERSIR compounds to ansiRNA targeted to an mRNA. In certain such embodiments, the target mRNAencodes a protein involved in metabolism. In certain such embodiments,the target mRNA encodes a protein involved in cardiac function. Incertain embodiments, the target mRNA encodes a protein involved inblood-clotting. Exemplary siRNA compounds targeting any of a variety oftarget proteins are known in the art. Further, methods for preparingsiRNA against a target gene are well known in the art and readilyavailable to one of skill in the art.

Without limitations, target genes for siRNAs include, but are notlimited to genes promoting unwanted cell proliferation, growth factorgene, growth factor receptor gene, genes expressing kinases, an adaptorprotein gene, a gene encoding a G protein super family molecule, a geneencoding a transcription factor, a gene which mediates angiogenesis, aviral gene, a gene required for viral replication, a cellular gene whichmediates viral function, a gene of a bacterial pathogen, a gene of anamoebic pathogen, a gene of a parasitic pathogen, a gene of a fungalpathogen, a gene which mediates an unwanted immune response, a genewhich mediates the processing of pain, a gene which mediates aneurological disease, an allene gene found in cells characterized byloss of heterozygosity, or one allege gene of a polymorphic gene.

Specific exemplary target genes for the siRNAs include, but are notlimited to, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF beta gene;Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS gene; MEKK gene; JNKgene; RAF gene; Erk1/2 gene; PCNA(p21) gene; MYB gene; c-MYC gene; JUNgene; FOS gene; BCL-2 gene; Cyclin D gene; VEGF gene; EGFR gene; CyclinA gene; Cyclin E gene; WNT-1 gene; beta-catenin gene; c-MET gene; PKCgene; NFKB gene; STAT3 gene; survivin gene; Her2/Neu gene; topoisomeraseI gene; topoisomerase II alpha gene; p73 gene; p21(WAF1/CIP1) gene,p27(KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumorsuppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressorgene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9,BCR/ABL fusion gene; TEL/AML1 fusion gene; EWS/FLI1 fusion gene;TLS/FUS1 fusion gene; PAX3/FKHR fusion gene; AML1/ETO fusion gene; alphav-integrin gene; Flt-1 receptor gene; tubulin gene; Human PapillomaVirus gene, a gene required for Human Papilloma Virus replication, HumanImmunodeficiency Virus gene, a gene required for Human ImmunodeficiencyVirus replication, Hepatitis A Virus gene, a gene required for HepatitisA Virus replication, Hepatitis B Virus gene, a gene required forHepatitis B Virus replication, Hepatitis C Virus gene, a gene requiredfor Hepatitis C Virus replication, Hepatitis D Virus gene, a generequired for Hepatitis D Virus replication, Hepatitis E Virus gene, agene required for Hepatitis E Virus replication, Hepatitis F Virus gene,a gene required for Hepatitis F Virus replication, Hepatitis G Virusgene, a gene required for Hepatitis G Virus replication, Hepatitis HVirus gene, a gene required for Hepatitis H Virus replication,Respiratory Syncytial Virus gene, a gene that is required forRespiratory Syncytial Virus replication, Herpes Simplex Virus gene, agene that is required for Herpes Simplex Virus replication, herpesCytomegalovirus gene, a gene that is required for herpes Cytomegalovirusreplication, herpes Epstein Barr Virus gene, a gene that is required forherpes Epstein Barr Virus replication, Kaposi's Sarcoma-associatedHerpes Virus gene, a gene that is required for Kaposi'sSarcoma-associated Herpes Virus replication, JC Virus gene, human genethat is required for JC Virus replication, myxovirus gene, a gene thatis required for myxovirus gene replication, rhinovirus gene, a gene thatis required for rhinovirus replication, coronavirus gene, a gene that isrequired for coronavirus replication, West Nile Virus gene, a gene thatis required for West Nile Virus replication, St. Louis Encephalitisgene, a gene that is required for St. Louis Encephalitis replication,Tick-borne encephalitis virus gene, a gene that is required forTick-borne encephalitis virus replication, Murray Valley encephalitisvirus gene, a gene that is required for Murray Valley encephalitis virusreplication, dengue virus gene, a gene that is required for dengue virusgene replication, Simian Virus 40 gene, a gene that is required forSimian Virus 40 replication, Human T Cell Lymphotropic Virus gene, agene that is required for Human T Cell Lymphotropic Virus replication,Moloney-Murine Leukemia Virus gene, a gene that is required forMoloney-Murine Leukemia Virus replication, encephalomyocarditis virusgene, a gene that is required for encephalomyocarditis virusreplication, measles virus gene, a gene that is required for measlesvirus replication, Vericella zoster virus gene, a gene that is requiredfor Vericella zoster virus replication, adenovirus gene, a gene that isrequired for adenovirus replication, yellow fever virus gene, a genethat is required for yellow fever virus replication, poliovirus gene, agene that is required for poliovirus replication, poxvirus gene, a genethat is required for poxvirus replication, plasmodium gene, a gene thatis required for plasmodium gene replication, Mycobacterium ulceransgene, a gene that is required for Mycobacterium ulcerans replication,Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, agene that is required for Mycobacterium leprae replication,Staphylococcus aureus gene, a gene that is required for Staphylococcusaureus replication, Streptococcus pneumoniae gene, a gene that isrequired for Streptococcus pneumoniae replication, Streptococcuspyogenes gene, a gene that is required for Streptococcus pyogenesreplication, Chlamydia pneumoniae gene, a gene that is required forChlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in loss of heterozygosity (LOH) cells, oneallele gene of a polymorphic gene and combinations thereof.

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in duploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific modulation ofone allele of an essential gene with a composition of the invention.

In certain embodiments, the invention provides REVERSIR compound to ansiRNA that modulates a micro-RNA.

REVERSIR compounds are oligomeric compounds. Accordingly, in certainembodiments, REVERSIR compounds comprise, for example and withoutlimitation, any of the modifications and motifs described in thediscussion herein for oligomeric compounds.

In certain embodiments, motifs are designed with consideration given toboth the siRNA and the REVERSIR compound. In certain embodiments, aREVERSIR compound could comprise 4 or more contiguous DNA-like monomers.In certain embodiments, the resulting RNA/DNA duplex could activateRNase H, resulting in cleavage of the RNA-like antisense compound. Incertain embodiments, REVERSIR activity does not depend on enzymaticactivity. In certain such embodiments, compounds designed without regardfor enzymatic compatibility may incorporate modifications to improveother attributes. For example, certain motifs yield oligomeric compoundswith high affinity for a target nucleic acid, but that are unable toelicit enzymatic cleavage of that target. Such motifs may be useful forREVERSIR compounds in embodiments where cleavage of the siRNA is notnecessary.

In certain embodiments, one strand of the siRNA, e.g., the strandcomplementary to REVERSIR compound, and corresponding REVERSIR compoundare the same length. In some embodiments, one strand of the siRNA, e.g.,the strand complementary to REVERSIR compound, and correspondingREVERSIR compound are different lengths. In some embodiments, theREVERSIR compound is shorter than the corresponding complementary strandfrom the siRNA. In some embodiments, the REVERSIR compound is shorter by1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides than the correspondingcomplementary strand from the siRNA.

In certain embodiments, antisense strand of the siRNA and correspondingREVERSIR compound are the same length. In some embodiments, antisensestrand of the siRNA and corresponding REVERSIR compound are differentlengths. In some embodiments, the REVERSIR compound is shorter than thecorresponding complementary antisense strand from the siRNA. In someembodiments, the REVERSIR compound is shorter by 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more nucleotides than the corresponding complementary antisensestrand from the siRNA.

In certain embodiments, sense strand of the siRNA and correspondingREVERSIR compound are the same length. In some embodiments, sense strandof the siRNA and corresponding REVERSIR compound are different lengths.In some embodiments, the REVERSIR compound is shorter than thecorresponding complementary sense strand from the siRNA. In someembodiments, the REVERSIR compound is shorter by 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more nucleotides than the corresponding complementary sensestrand from the siRNA.

In certain embodiments, an siRNA and a REVERSIR compound areadministered to a patient. In certain such embodiments, pharmaceuticalcompositions comprising an siRNA and those comprising a REVERSIRcompound comprise the same formulation. In certain embodiments,pharmaceutical compositions comprising an siRNA and those comprising aREVERSIR compound comprise different formulations. In certainembodiments an siRNA and a REVERSIR compound are administered by thesame route. In certain embodiments an siRNA and a REVERSIR compound areadministered by different routes. For example, in certain embodiments,an siRNA is administered orally and a REVERSIR compound is administeredby injection. In certain embodiments, the dosages of the siRNA and theREVERSIR compound are the same. In certain embodiments, the dosages ofthe siRNA and the REVERSIR compound are different.

In certain embodiments, the toxicity profiles of the siRNA and theREVERSIR compound are similar. In certain embodiments, such toxicityprofiles are different. For example, in certain embodiments, an siRNAcan be intended for chronic administration and the REVERSIR compound isonly intended for acute use as needed. In such embodiments, thetolerance for toxic side-effects of the REVERSIR compound can be higher.Accordingly, modifications and motifs that may be too toxic for use inan siRNA can be acceptable in a REVERSIR compound. For example, incertain embodiments, oligomeric compounds comprising one or more LNAnucleoside have been shown to have high affinity for a target nucleicacid, but in certain embodiments have been shown to cause toxicity atrelatively low concentrations. For certain siRNAs, where chronicadministration is intended, certain such compounds comprising LNA maynot be suitable. However, in embodiments where a REVERSIR compound isnot intended for chronic administration, but rather for acuteadministration when siRNA activity is problematic, such LNAmodifications in an antidote compound are acceptable. The increasedaffinity of LNA can improve the REVERSIR effect and since the REVERSIRcompound is only administered for a short period of time, and possiblywhen the patient is in distress, the increased toxicity of LNA may bejustified. Other high affinity, but potentially toxic modifications arealso known.

In certain embodiments, activity of siRNA is counteracted by anon-oligomeric REVERSIR. For example, in certain embodiments, when thetarget nucleic acid is a target mRNA encoding a protein it is desirableto reduce the activity of siRNA and to increase in the amount of thetarget protein (e.g., target protein amount has gone too low, orcircumstances have changed resulting in the desire to restore targetprotein amount). In such embodiments, one can simply administer thetarget protein itself. Such administration will immediately reverse thesiRNA activity of target protein reduction. However, it can also bedesirable to administer an oligomeric REVERSIR compound according to thepresent invention. For example, the target protein may have a shorthalf-life in the animal. Accordingly, to maintain the restored targetprotein concentration would require repeated administration of targetprotein until the siRNA has cleared and normal protein expression isrestored. In certain such embodiments, it is still desirable toadminister an REVERSIR compound to shorten the duration of the siRNAactivity. In certain embodiments an oligomeric REVERSIR compound isco-administered with a non-oligomeric REVERSIR. In certain suchembodiments, the non-oligomeric REVERSIR is a target protein. In certainembodiments, the non-oligomeric REVERSIR compound is a protein havingsimilar physiological effect as a target protein or that stimulatesexpression of the target protein.

Research Tools

In certain instances, siRNAs have been used as research tools. Forexample, researchers investigating the function of a particular geneproduct can design siRNAs to reduce the amount of that gene productpresent in a cell or an animal and observe phenotypic changes in thecell or animal. In certain embodiments, the present invention providesmethods for reducing the amount of a gene product in a cell or animalthrough RNAi and then reducing that RNAi activity, thereby restoring theinhibited gene product. In certain embodiments, investigators can usesuch techniques to characterize proteins or untranslated nucleic acids.In certain embodiments, investigators can vary the amount of timebetween siRNA and REVERSIR compounds administration. In certainembodiments, such experiments are used to investigate kinetics and/orturnover of gene products and/or certain cellular functions.

As described herein, the invention provides methods comprisingadministering to a subject a siRNA followed by administering a REVERSIRcompound or composition comprising same. Without limitation, the siRNAand the REVERSIR compound can be conjugated or unconjugated. Further,the siRNA and the REVERSIR compound can be independently encapsulated ina lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.Moreover, the siRNA and the REVERSIR compound can be administered,independently, via any appropriate route or mode of administration. Forexample, the siRNA and the REVERSIR compound can be independentlyadministered via intravenous administration (IV) or via subcutaneousadministration (SC).

Accordingly, in some embodiments, the invention provides methodscomprising administering to a subject an unconjugated siRNA followed byadministering a conjugated REVERSIR compound, wherein the REVERSIRcompound is encapsulated in a lipid formulation, e.g., a LNP, or othernucleic acid-lipid particle, and wherein the REVERSIR compound isadministered via intravenous administration.

In some other embodiments, the invention provides methods comprisingadministering to a subject an unconjugated siRNA followed byadministering a conjugated REVERSIR compound, wherein the REVERSIRcompound is encapsulated in a lipid formulation and the REVERSIRcompound is administered via subcutaneous administration.

In yet some other embodiments, the invention provides methods comprisingadministering to a subject a conjugated siRNA followed by administeringa conjugated REVERSIR compound, wherein the REVERSIR compound isencapsulated in a lipid formulation and the REVERSIR compound isadministered via intravenous administration.

In still some other embodiments, the invention provides methodscomprising administering to a subject a conjugated siRNA followed byadministering a conjugated REVERSIR compound, wherein the REVERSIRcompound is encapsulated in a lipid formulation and the REVERSIRcompound is administered via subcutaneous administration.

While the above described embodiments specify conjugated REVERSIRcompounds, unconjugated REVERSIR compounds can also be used. Thus, insome embodiments, the invention provides methods comprisingadministering to a subject an unconjugated siRNA followed byadministering an unconjugated REVERSIR compound, wherein the REVERSIRcompound is encapsulated in a lipid formulation and the REVERSIRcompound is administered via intravenous administration. In some otherembodiments, the invention provides methods comprising administering toa subject an unconjugated siRNA followed by administering anunconjugated REVERSIR compound, wherein the REVERSIR compound isencapsulated in a lipid formulation and the REVERSIR compound isadministered via subcutaneous administration. In yet some otherembodiments, the invention provides methods comprising administering toa subject a conjugated siRNA followed by administering an unconjugatedREVERSIR compound, wherein the REVERSIR compound is encapsulated in alipid formulation and the REVERSIR compound is administered viaintravenous administration.

In still some other embodiments, the invention provides methodscomprising administering to a subject a conjugated siRNA followed byadministering an unconjugated REVERSIR compound, wherein the REVERSIRcompound is encapsulated in a lipid formulation and the REVERSIRcompound is administered via subcutaneous administration.

Kits

In certain embodiments, the present invention provides kits comprisingone or more siRNAs and one or more corresponding REVERSIR compound. Incertain embodiments, such kits are intended for therapeutic application.In certain embodiments, such kits are intended for research use.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

The nucleoside sequences set forth in the sequence listing and Examples,are independent of any modification to a sugar moiety, a monomericlinkage, or a nucleobase. As such, oligomeric compounds defined by a SEQID NO can comprise, independently, one or more modifications to a sugarmoiety, an internucleoside linkage, or a nucleobase.

EXAMPLES Example 1: Reversal of Antithrombin (AT) Knockdown in Wild-TypeMice

Sixty wild-type mice (C57BL/6, female) were bled on Day −1 to obtainpre-dose blood samples. All animals were subsequently injectedsubcutaneously with a single dose of ALN-57213 at 3 mg/kg on Day 0. OnDay 3, 3 mice per group received a single subcutaneous injection of oneof 19 different reversal agents (Table 2) at a dose of 10 mg/kg. Threeanimals did not receive an injection on Day 3 and served as an untreatedcontrol. All animals were bled on Days 7, 11, and 15 to obtain serumsamples. Serum samples were then analyzed for AT antigen level by ATELISA and were normalized to the pre-dose AT level for each animal. FIG.1 displays normalized group mean (±S.D.) AT levels. As indicated in FIG.1, multiple reversal agents reduced the level of AT knockdown mediatedby a single subcutaneous dose of ALN-57213.

TABLE 2Design and Synthesis of 19 exemplary REVERSIR compounds (primarily varying length and LNAs)SEQ Design ID length feature REVERSIR sequence Alnylam # NO (nt) #LNAsDescription Basic cugguuaacaccauuuacuucadAL96 A-132289 1 22 0Full length 1-22; all design OMe; No PS linkagescsusgguuaacaccauuuacuuscsadAL96 A-132290 2 22 0 Full length 1-22; allOMe; two PS linkages each at both ends Lengthcsusgsgsususasascsascscsasususu A-132291 3 22 0 Full length 1-22; allreduction sascsususcsadAL96 OMe gsususasascsascscsasusususascsu A-1322924 18 0 Fragment 4-22; all suscsadAL96 OMeascsascscsasusususascsususcsadAL96 A-132293 5 15 0 Fragment 8-22; allOMe LNAs in cs(Tlns)gsgsususasascsascscsasu A-132294 6 22 2Full length 1-22; all full sususascsususcs(Alns)adAL96 OMe except LNAlength; @ n2, 22 22-mer cs(Tlns)gs(Glns)ususasascsascscsa A-132295 7 224 Full length 1-22; all susususascsus(Tlns)cs(Alns)adAL96 OMe except LNA@ n2, 4, 20, 22 cs(Tlns)gs(Glns)ususasascsascscsa A-132296 8 22 5Full length 1-22; all susususas(m5clns)us(Tlns)cs(Alns) OMe except LNAadAL96 @ n2, 4, 18, 20, 22 cs(Tlns)gs(Glns)ususasascs(Alns) A-132297 922 6 Full length 1-22; all cscsasusususas(m5clns)us(Tlns)csOMe except LNA @ n2, (Aln)dAL96 4, 10, 18, 20, 22 LNAs in(Glns)ususasascsascscsasusususas A-132298 10 18 2 Fragment 4-22; all4-22 csususcs(Aln)dAL96 OMe except LNA fragment; @ n4, 22 18-mer(Glns)ususasascsascscsasusususas A-132299 11 18 3 Fragment 4-22; allcsus(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 20, 22(Glns)ususasascsascscsasusususas A-132300 12 18 4 Fragment 4-22; all(m5clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 18, 20, 22(Glns)ususasascs(Alns)cscsasusu A-132301 13 18 5 Fragment 4-22; allsusas(m5clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ n4, 10, 18, 20, 22LNAs in (Alns)csascscsasusususascsususcs A-132302 14 15 2Fragment 8-22; all 8-22 (Aln)dAL96 OMe except LNA fragment; @ n8, 2215-mer (Alns)(m5clns)ascscsasusususascsus A-132303 15 15 4Fragment 8-22; all (Tlns)cs(Aln)dAL96 OMe except LNA @ n8, 9, 20, 22(Alns)cs(Alns)cscsasusususas A-132304 16 15 5 Fragment 8-22; all(m5Clns)us(Tlns)cs(Aln)dAL96 OMe except LNA @ 8, 10, 18, 20, 22 DNA ntscs(Tlns)dGs(Glns)ususasascs(Alns)dCscsa A-132305 17 22 6Full length; LNA in LNA susususas(m5clns)dTs(Tlns)dCs(Aln)dAL96@ n2, 4, 10, 18, 20, region; 22; DNA @ 3, 11, varying 19, 21 length(Glns)ususasascs(Alns)dCscsasusususas A-132306 18 22 5Fragment 4-22; all (m5clns) dTs(Tlns)dCs(Aln)dAL96 OMe except LNA@ n4, 10, 18, 20, 22; DNA @ 11, 19, 21(Alns)cs(Alns)dCscsasusususas(m5clns)dTs A-132307 19 22 5Fragment 8-22; all (Tlns)dCs(Aln)dAL96 OMe except LNA @ 8, 10, 18, 20,22; DNA @ 11, 19, 21 If, not indicated, all OMe with full PS linkages

TABLE 3 Inclusion of DNA residues may reduce activity Serum AT levelsSEQ length # Day Day Day ID REVERSIR sequence (nt) LNAs 7 11 15 Alnylam# NO cs(Tlns)gs(Glns)ususasascs(Alns)cscsasusususas 22 6 0.92 1.06 1.03A-132297 9 (m5clns)us(Tlns)cs(Aln)dAL96cs(Tlns)dGs(Glns)ususasascs(Alns)dCscsasusususas 22 6 0.86 1.00 0.97A-132305 17 (m5clns)dTs(Tlns)dCs(Aln)dAL96(Glns)ususasascs(Alns)cscsasusususas(m5clns)us 18 5 0.85 0.98 1.01A-132301 13 (Tlns)cs(Aln)dAL96(Glns)ususasascs(Alns)dCscsasusususas(m5clns) 18 5 0.61 0.66 0.68A-132306 18 dTs(Tlns)dCs(Aln)dAL96(Alns)cs(Alns)cscsasusususas(m5Clns)us(Tlns)cs 15 5 1.00 0.80 0.91A-132304 16 (Aln)dAL96 (Alns)cs(Alns)dCscsasusususas(m5clns)dTs(Tlns) 155 0.88 0.77 0.72 A-132307 19 dCs(Aln)dAL96

TABLE 4 In vitro Transfection of REVERSIR compounds targeting AD-57213In vitro dual-luc-2pt siRNA then REVERSIR dose response REVERSIR thensiRNA 1 10 transfection transfection REVERSIR nM nM IC₅₀ IC₅₀ ID avg SDavg SD (nM) (nM) A-132289 21.1 1.4 40.5 5.6 0.786 0.650 A-132290 41.55.5 77.0 7.2 0.569 0.955 A-132291 24.4 1.6 46.5 6.1 0.965 0.749 A-13229224.6 1.8 47.2 7.9 0.498 0.341 A-132293 16.3 0.9 17.5 2.0 0.119 N/AA-132294 30.6 6.0 61.3 3.2 0.322 0.440 A-132295 45.3 5.2 82.3 6.0 0.4080.132 A-132296 50.4 6.0 79.3 5.3 0.381 0.053 A-132297 54.8 16.6 83.4 4.60.355 0.061 A-132298 24.1 3.3 56.0 4.0 A-132299 26.7 4.4 56.6 4.0A-132300 40.3 5.9 70.1 1.6 A-132301 69.7 6.9 88.4 8.7 0.383 0.075A-132302 18.0 2.7 31.7 2.7 A-132303 17.8 1.0 45.4 8.8 A-132304 25.1 3.357.1 7.3 1.060 0.382 A-132305 48.6 3.8 75.8 10.0 A-132306 53.4 4.0 72.27.4 A-132307 18.2 1.6 46.1 9.4

TABLE 54-dose free-uptake in vitro of REVERSIR compounds targeting AD-57213 SEQ100 10 1 0.1 ID nM nM nM nM REVERSIR NO 5′-Sequence-3′ avg SD avg SD avgSD avg SD A-132301 13 (Glns)ususasascs(Alns)cscsasusususas 76.9 5.9 61.55.8 19.6 16.7 3.8 1.3 (m5Clns)us(Tlns)cs(Aln)dAL96 A-135676 20(Glns)ususasascs(Alns)csgsususususas 2.9 0.7 7.4 4.2 2.8 2.0 3.8 1.1(Glns)as(Tlns)cs(Aln)dAL96 A-135677 21(Alns)cscsususas(Tlns)asusascsgsasus 3.5 2.1 6.4 1.9 2.5 1.2 4.7 1.0(Tlns)as(m5Clns)us(m5Cln)dAL96 A-135678 22gs(Tces)usasascsas(m5Cces)csasusususas 80.4 16.9 104.6 23.4 35.0 27.513.4 3.4 (m5Cces)us(Tces)(m5Cces)adAL96 A-135679 23gs(Tlns)usasascsas(m5Clns)csasusususas 104.9 19.1 97.4 32.9 37.9 16.520.5 5.5 (m5Clns)us(Tlns)(m5Clns)adAL96 A-135680 24gs(Tces)usasascsas(m5Cces)csausususas 85.9 16.1 128.3 18.2 27.8 23.210.5 4.6 (m5Cces)us(Tces)(m5Cces)adAL96 A-135681 25gs(Tces)usasascsas(m5Cces)csdAusususas 88.0 29.1 137.6 30.3 36.3 21.113.8 6.0 (m5Cces)us(Tces)(m5Cces)adAL96 A-135682 26gsususasascsascscsas(Tces)us(Tces)as 82.8 17.0 129.2 47.8 31.6 23.0 11.64.7 (m5Cces)us(Tces)(m5Cces)adAL96 A-135683 27gsususasascsascscsas(Tlns)us(Tlns)as 97.5 22.4 100.5 25.6 41.2 19.2 14.64.4 (m5Clns)us(Tlns)(m5Clns)adAL96 A-135684 28gs(Tces)(Tces)asas(m5Cces)as(m5Cces) 72.4 23.5 95.8 40.8 7.6 0.9 6.4 0.9(m5Cces)asusususascsususcsadAL96 A-135685 29gs(Tlns)(Tlns)asas(m5Clns)as(m5Clns) 67.7 3.4 84.2 7.8 14.2 5.8 5.0 3.0(m5Clns)asusususascsususcsadAL96 A-135686 30gsususasascsas(m5Cces)(m5Cces)as(Tces) 64.8 13.6 115.7 34.8 30.3 25.84.3 1.8 (Tces)(Tces)ascsususcsadAL96 A-135687 31gsususasascsas(m5Clns)(m5Clns)as(Tlns) 69.1 12.9 42.4 15.9 14.0 4.5 2.91.1 (Tlns)(Tlns)ascsususcsadAL96 A-135688 32gs(Tces)usasas(m5Cces)ascscsas(Tces) 87.6 16.5 44.4 13.7 22.5 2.9 7.13.7 ususas(m5Cces)usus(m5Cces)adAL96 A-135689 33gs(Tlns)usasas(m5Clns)ascscsas(Tlns) 95.8 20.5 102.5 20.0 53.3 22.3 16.77.9 ususas(m5Clns)usus(m5Clns)adAL96 A-135690 34gsususasas(m5Cces)as(m5Cces)(m5Cces) 96.7 16.9 66.7 25.9 29.1 3.8 13.21.0 asusususas(m5Cces)usus(m5Cces)adAL96 A-135691 35gsususasas(m5Clns)as(m5Clns)(m5Clns) 125.8 27.0 93.7 52.2 36.7 21.4 15.811.2 asusususas(m5Clns)usus(m5Clns)adAL96 A-135692 36gsususasas(m5Cces)ascs(m5Cces)asusus 110.9 16.0 106.8 20.1 15.6 6.0 11.04.0 usas(m5Cces)usus(m5Cces)adAL96 A-135693 37gsususasas(m5Clns)ascs(m5Clns)asusus 118.1 15.3 93.2 40.3 62.6 33.9 19.47.2 usas(m5Clns)usus(m5Clns)adAL96 A-135694 38gsususasas(m5Cces)ascs(m5Cces)as(Tces) 85.4 9.1 69.7 15.1 22.1 13.4 11.13.0 ususascsusus(m5Cces)adAL96 A-135695 39gsususasascsascs(m5Cces)as(Tces)ususas 66.3 11.3 56.3 19.4 14.9 9.3 8.02.1 csus(Tces)(m5Cces)adAL96 A-135696 40gsususasascsascscsas(Tces)us(Tces)ascs 80.5 15.7 96.0 23.2 18.6 3.3 8.52.7 (Tces)us(m5Cces)adAL96 A-135697 41gsususasascsascscsas(Tces)ususascs 32.6 6.8 47.5 10.0 8.4 4.4 3.4 1.5(Tces)(Tces)(m5Cces)adAL96 A-135698 42gs(Tces)usasas(m5Cces)ascs(m5Cces)as 83.2 36.7 37.4 10.0 14.5 5.7 4.32.4 (Tces)ususascsususcsadAL96 A-135699 43gs(Tces)(Tces)asas(m5Cces)ascscsas 56.3 10.4 40.3 21.7 9.1 4.7 2.6 1.1(Tces)ususascsususcsadAL96 A-135700 44gsususasascsascscsas(Tces)ususascs 56.3 15.9 46.5 24.4 9.4 5.8 5.8 1.0(Tces)us(m5Cces)adAL96 A-135701 45 gs(Tces)usasas(m5Cces)ascscsas(Tces)90.1 8.3 68.3 12.3 14.3 11.9 12.5 4.9 ususascsususcsadAL96 A-135702 46gs(Tces)(Tces)asascsascscsas(Tces) 68.9 11.9 43.7 24.4 9.5 3.9 13.1 3.3ususascsususcsadAL96 A-135703 47 gs(Tces)(Tces)asas(m5Cces) 57.7 7.738.9 16.9 11.1 5.3 14.8 1.0 ascscsasusususascsususcsadAL96 A-135704 48gs(Tces)usaaca(m5Cce)casusuusa 108.7 17.2 80.6 20.4 52.4 31.7 23.2 5.6(m5Cce)us(Tce)(m5Cces)adAL96 A-135705 49gs(Tce)usaaca(m5Cce)casuuua(m5Cce) 94.4 19.0 89.6 7.5 49.1 21.6 21.8 3.5us(Tce)(m5Cce)adAL96 A-135706 50 gs(Tces)uaaca(m5Cce)cauuua(m5Cce) 69.57.7 52.9 13.6 20.2 6.2 11.9 3.8 u(Tces)(m5Cces)adAL96 A-135707 51gs(Tce)uaaca(m5Cce)cauuua(m5Cce) 71.1 19.5 58.7 10.0 28.3 23.8 14.2 4.6u(Tce)(m5Cces)adAL96 A-135708 52 gs(Tces)uaacaccauuuacuuscsadAL96 44.57.6 47.2 27.9 11.4 4.4 8.6 4.9 A-135709 53gsus(Tce)aacaccauuuacuuscsadAL96 33.2 6.1 43.8 17.3 7.4 3.6 6.1 1.6A-135710 54 gsusuaa(m5Cce)accauuuacuuscsadAL96 34.8 15.8 67.0 9.7 16.48.3 5.7 1.8 A-135711 55 gsusuaaca(m5Cce)cauuuacuuscsadAL96 66.8 15.629.9 18.5 4.2 1.1 3.6 1.5 A-135712 56 gsusuaacac(m5Cce)auuuacuuscsadAL9643.0 11.0 35.6 11.4 9.6 5.1 6.6 2.8 A-135713 57gsusuaacacca(Tce)uuacuuscsadAL96 48.8 7.2 39.1 6.3 14.7 6.2 8.4 2.8A-135714 58 gsusuaacaccau(Tce)uacuuscsadAL96 44.1 17.1 32.0 13.4 20.712.1 9.5 2.2 A-135715 59 gsusuaacaccauu(Tce)acuuscsadAL96 51.7 13.8 42.620.5 19.7 13.9 8.7 4.5 A-135716 60 gsusuaacaccauuua(m5Cce)uuscsadAL9684.0 8.2 73.9 36.3 27.0 21.5 14.9 7.8 A-135717 61gsusuaacaccauuuac(Tce)uscsadAL96 61.5 20.7 41.9 5.5 13.9 8.1 7.7 2.8A-135718 62 gsusuaacaccauuuacu(Tces)csadAL96 51.8 13.2 33.1 13.9 7.4 1.57.4 2.6 A-135719 63 gsusuaacaccauuuacuus(m5Cces)adAL96 61.0 12.9 38.14.4 13.6 7.8 9.9 4.8 A-135720 64 gs(Tlns)uaacaccauuuacuuscsadAL96 61.310.7 43.5 14.8 16.2 12.4 5.6 2.4 A-135721 65gsus(Tln)aacaccauuuacuuscsadAL96 35.4 7.5 40.8 17.9 9.1 4.2 5.2 2.0A-135722 66 gsusuaa(m5Cln)accauuuacuuscsadAL96 24.6 13.4 38.8 11.2 7.91.7 3.6 1.6 A-135723 67 gsusuaaca(m5Cln)cauuuacuuscsadAL96 47.8 15.517.6 4.3 4.4 1.0 2.0 1.0 A-135724 68 gsusuaacac(m5Cln)auuuacuuscsadAL9656.4 4.0 18.6 7.0 8.6 5.9 4.6 3.1 A-135725 69gsusuaacacca(Tln)uuacuuscsadAL96 55.1 13.9 22.8 7.1 14.2 3.5 8.3 3.4A-135726 70 gsusuaacaccau(Tln)uacuuscsadAL96 39.8 4.3 23.1 9.4 6.3 2.910.2 4.4 A-135727 71 gsusuaacaccauu(Tln)acuuscsadAL96 54.4 15.0 25.3 5.612.0 4.8 8.8 3.3 A-135728 72 gsusuaacaccauuua(m5Cln)uuscsadAL96 89.132.8 45.7 18.9 27.5 4.5 18.1 4.8 A-135729 73gsusuaacaccauuuac(Tln)uscsadAL96 57.7 21.0 32.3 18.0 11.4 5.6 9.0 2.3A-135730 74 gsusuaacaccauuuacu(Tlns)csadAL96 54.9 10.3 25.2 8.9 20.616.3 11.1 3.8 A-135731 75 gsusuaacaccauuuacuus(m5Clns)adAL96 58.2 8.428.8 8.1 10.1 7.0 9.8 1.7 A-135732 76gsususasasY5sascscsasusususascsusus 72.6 4.5 39.3 17.7 7.5 1.9 6.6 2.7Y5sadAL96 A-135733 77 gsususasasY5sascscsasusususasY5susu 54.3 10.2 30.220.7 3.4 2.2 5.6 4.0 scsadAL96 A-135734 78gsususasascsasY5scsasusususascsusus 55.4 31.8 43.9 25.4 10.8 4.8 3.0 0.4Y5sadAL96 A-135735 79 gsususasascsascsY5sasusususasY5susu 65.1 10.4 27.014.7 5.1 1.7 2.5 1.4 scsadAL96 A-135736 80gsususasasY5sasY5scsasusususascsusu 64.8 31.1 17.1 12.4 4.5 2.7 3.7 1.1scsadAL96 A-135737 81 gsususasasY5sascsY5sasusususascsusu 64.3 20.4 26.915.7 4.7 2.3 4.6 2.0 scsadAL96 A-135738 82gsususasascsascscsasusususasY5susus 60.5 20.5 25.2 12.3 8.9 3.8 7.2 4.0Y5sadAL96 A-135739 83 gsususasasY5sascscsasusususascsusus 76.9 12.2 30.315.6 11.5 5.7 7.4 2.4 csadAL96 A-135740 84gsususasascsasY5scsasusususascsusus 99.1 38.2 43.2 20.0 9.4 6.0 8.4 4.4csadAL96 A-135741 85 gsususasascsascsY5sasusususascsusus 86.5 12.9 55.133.0 7.2 1.3 5.1 3.0 csadAL96 A-135742 86gsususasascsascscsasusususasY5susus 81.2 10.4 33.4 17.8 13.4 3.8 9.6 2.4csadAL96 A-135743 87 gsususasascsascscsasusususascsususY 69.0 13.7 36.620.8 15.3 16.7 7.5 3.5 5sadAL96 A-135744 88gsususasasY24sascsY24sasusususascsu 30.1 6.3 19.7 6.2 7.8 3.1 5.0 1.8susY24sadAL96 A-135745 89 gsususasasY24sascscsasusususasY24su 9.6 2.88.7 5.6 5.8 3.1 3.0 0.8 susY24sadAL96 A-135746 90gsususasasY24sascscsasusususascsusu 6.7 2.9 11.9 7.8 2.8 1.2 2.5 1.2sY24sadAL96 A-135747 91 gsususasasY24sascscsasusususasY24su 22.8 11.85.1 1.7 2.0 0.7 2.0 0.6 suscsadAL96 A-135748 92gsususasascsascsY24sasusususasY24su 28.3 1.9 7.0 3.2 2.9 2.3 4.2 1.5suscsadAL96 A-135749 93 gsususasasY24sasY24scsasusususascsu 24.1 6.613.6 4.4 4.9 2.3 3.5 2.0 suscsadAL96 A-135750 94gsususasascsascscsasusususasY24susu 16.7 5.1 12.7 10.0 9.9 10.1 6.7 4.3sY24sadAL96 A-135751 95 gsususasasY27sascscsasusususasY27 60.8 23.5 16.67.7 8.6 5.9 4.8 2.6 sususY27sadAL96 A-135752 96gsusuaacaccauuuacuuscsadAL96 58.1 29.6 32.3 17.0 14.7 12.5 7.6 3.4A-135753 97 gsUfsusAfsasCfsasCfscsAfsusUf 48.0 10.1 23.0 12.4 8.0 4.43.6 2.8 susAfscsUfsusCfsadAL96 A-135754 98 GfsusUfsasAfscsAfscscsasUfsus84.3 18.9 29.3 14.2 12.7 9.2 5.8 2.6 UfsasCfsusUfscsAfdAL96 A-135755 99gsUfsusAfsasCfsasCfscsasusUfsus 32.9 6.8 15.4 4.0 7.5 5.4 5.3 1.9AfscsUfsusCfsadAL96 A-135756 100 gsUfsuAfsaCfsaCfscasuUfsuAfscUfsus 42.413.7 29.1 13.4 4.9 3.7 6.3 3.7 CfsadAL96 A-135757 101gs(Tces)usAfsas(m5Cces)asCfscsasus 44.3 12.7 20.6 5.1 4.2 3.7 2.9 1.7UfsusAfscsUfsusCfsadAL96 A-135758 102gsUfsusAfsasCfsasCfscsasusUfsusAfscs 27.4 9.7 20.4 11.6 12.1 8.0 2.8 3.5(Tces)us(m5Cces)adAL96 A-135759 103 gsUfsusAfsasCfsasCfs(m5Cces)as 68.315.7 26.9 11.7 4.1 1.1 2.4 0.3 (Tces)UfsusAfscsUfsusCfsadAL96 A-135760104 gs(Tces)usAfsas(m5Cces)asCfscsasus 61.4 14.2 22.3 1.4 2.5 1.0 2.10.4 (Tces)usAfscsUfsusCfsadAL96 A-135761 105gsgs(Tces)usasascsas(m5Cces)csasusus 71.2 18.9 82.9 11.2 12.8 6.7 6.03.3 usas(m5Cces)us(Tces)(m5Cce)dAL96 A-135762 106(Tces)gsgs(Tces)usasascsas(m5Cces) 19.3 7.6 17.3 4.1 5.9 5.4 3.8 1.9csasusususas(m5Cces)us(Tce)dCL96 A-135763 107cs(Tces)gsgs(Tces)usasascsas(m5Cces) 6.1 3.5 7.9 4.5 3.7 3.1 5.3 3.0csasusus(Tces)as(m5Cces)udTL96 A-135764 108gs(Tces)UfsAfsAfsCfsAfs(m5Cces)CfsAfsUfs 82.6 8.4 47.2 32.8 6.5 3.3 5.62.7 UfsUfsAfs(m5Cces)Ufs(Tces)(m5Cces)adAL96 A-135765 109gsUfsUfsAfsAfsCfsAfsCfsCfsAfsUfsUfsUfsA 88.7 12.8 30.2 7.5 7.0 2.5 3.72.1 fsCfsUfsUfsCfsadAL96 A-135766 110gsUfsUfAfAfCfAfCfCfAfUfUfUfAfCfUfUfs 6.1 3.4 5.5 3.1 2.5 1.6 4.0 1.0CfsadAL96

TABLE 6IC₅₀ free-uptake in vitro of REVERSIR compounds targeting AD-57213REVERSIR SEQ ID ID NO 5′-Sequence-3′ IC₅₀ (nM) A-138952 111csusgsgsususasascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 >100A-135683 112gsususasascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 9.6A-138953 113 ascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL962.5 A-138954 114csascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.49 A-138955115 ascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.71 A-138956116 cscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.29 A-138957117 csas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.27 A-138958 118as(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 0.22 A-138959 119(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 <0.015

TABLE 7REVERSIR compounds targeting AD-57213 used in the in vivo experimentsSEQ ID siRNA NO 5′-Sequence-3′ AD-57213 120GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 121usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg REVERSIR ID 5′-Sequence-3′ A-1322935 ascsascscsasusususascsususcsadAL96 A-132302 14(Alns)csascscsasusususascsususcs(Aln)dAL96 A-132303 15(Alns)(m5Clns)ascscsasusususascsus(Tlns)cs(Aln)dAL96 A-132304 16(Alns)cs(Alns)cscsasusususas(m5Clns)us(Tlns)cs(Aln)dAL96 A-132296 8cs(Tlns)gs(Glns)ususasascsascscsasusususas(m5Clns)us(Tlns)cs(Alns)adAL96A-132301 13(Glns)ususasascs(Alns)cscsasusususas(m5Clns)us(Tlns)cs(Aln)dAL96A-135678 22gs(Tces)usasascsas(m5Cces)csasusususas(m5Cces)us(Tces)(m5Cces)adAL96A-135679 23gs(Tlns)usasascsas(m5Clns)csasusususas(m5Clns)us(Tlns)(m5Clns)adAL96A-135704 48 gs(Tces)usaaca(m5Cce)casusuusa(m5Cce)us(Tce)(m5Cces)adAL96A-138953 113 ascsascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96A-140335 122 (Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-138955 115ascscsas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-140336 123(Tlns)as(m5Clns)(Tlns)(Tlns)(m5Clns)adAL96 A-138957 117csas(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-138959 119(Tlns)us(Tlns)as(m5Clns)us(Tlns)(m5Clns)adAL96 A-140339 124csas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140340 125(Tlns)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140337 126ascsascscsas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96 A-140338 127ascscsas(Tln)us(Tln)as(m5Cln)us(Tln)(m5Clns)adAL96

TABLE 84-dose free-uptake in vitro of REVERSIR compounds targeting AD-66568 SEQ100 10 1 0.1 ID nM nM nM nM REVERSIR NO 5′-Sequence-3′ avg SD avg SD avgSD avg SD A-138962.1 128 ususcsasgsusascscsususasgsasgsususcscsas 47.612.3 50.2 6.3 23.1 4.8 15.7 3.6 csusdAL96 A-138963.1 129ususcsasgsusascscsususasgsasgsususcscsas 48.9 13.8 57.6 7.7 33.4 3.222.8 5.4 cs(Tln)dAL96 A-138964.1 130ususcsasgsusascscsususasgsasgsususcscsas 67.4 17.0 67.1 10.2 23.6 6.819.8 4.8 (m5Clns)(Tln)dAL96 A-138965.1 131ususcsasgsusascscsususasgsasgsususcs(m5Clns) 69.7 11.1 65.1 11.9 33.811.3 21.3 3.3 as(m5Clns)(Tln)dAL96 A-138966.1 132ususcsasgsusascscsususasgsasgsus(Tlns)cs 63.8 13.5 63.9 6.4 22.2 3.615.9 3.8 (m5Clns)as(m5Clns)(Tln)dAL96 A-138967.1 133ususcsasgsusascscsususasgsasgs(Tlns)(Tlns) 53.1 3.2 60.9 6.4 21.4 3.716.1 1.6 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138968.1 134ususcsasgsusascscsus(Tlns)asgsasgs(Tlns) 57.9 8.6 59.4 14.8 20.8 4.117.3 0.7 (Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138969.1 135ususcsasgsusas(m5Clns)csus(Tlns)asgsasgs 71.0 12.6 63.4 8.6 24.0 1.816.0 3.5 (Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138970.1 136uscsasgsusascscsususasgsasgs(Tlns)(Tlns) 55.5 7.0 43.3 5.8 20.3 3.2 18.12.7 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138971.1 137csasgsusascscsususasgsasgs(Tlns)(Tlns) 44.3 20.0 65.0 13.8 20.3 3.3 21.53.7 cs(m5Clns)as(m5Clns)(Tln)dAL96 A-138972.1 138asgsusascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 56.8 17.3 59.3 10.2 26.29.5 20.2 4.4 as(m5Clns)(Tln)dAL96 A-138973.1 139gsusascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 42.3 16.0 53.7 7.7 29.1 7.018.6 4.2 as(m5Clns)(Tln)dAL96 A-138974.1 140usascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 59.7 18.3 55.8 6.9 27.9 3.820.6 5.8 as(m5Clns)(Tln)dAL96 A-138975.1 141ascscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as 50.0 5.0 51.2 5.4 38. 6 12.322.4 2.0 (m5Clns)(Tln)dAL96 A-138976.1 142cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 82.6 17.8 65.4 13.931.9 3.5 20.4 3.7 (Tln)dAL96 A-138977.1 143csususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 74.4 7.8 60.4 9.4 27.86.9 17.8 4.1 (Tln)dAL96 A-138978.1 144ususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 77.0 6.5 62.5 10.7 22.6 2.419.8 5.0 (Tln)dAL96 A-138979.1 145usasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 73.7 15.1 57.6 8.8 31.4 8.217.6 2.4 (Tln)dAL96 A-138980.1 146asgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln) 72.2 12.7 61.0 4.9 32.57.4 16.8 2.7 dAL96 A-138981.1 147gsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 81.6 9.0 66.4 7.7 33.36.2 20.2 1.5 A-138982.1 148asgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 104.3 8.9 61.1 12.0 41.28.2 24.5 2.6 A-138983.1 149 gs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL9684.6 22.8 83.1 12.6 52.0 14.6 24.4 2.6 A-138984.1 150(Tlns)(Tlns)cs(m5Clns)as(m5Clns)(Tln)dAL96 54.2 14.0 55.9 3.5 22.7 4.019.4 3.1 A-138985.1 151 csususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns)52.1 14.5 53.9 4.1 19.2 7.9 18.4 1.3 (Tlns)adAL96 A-138976.2 152cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 68.6 26.2 48.6 4.1 29.06.2 24.1 3.4 (Tln)dAL96 A-138986.1 153ascscsus(Tlns)asgsasgs(Tlns)(Tlns)cs(m5Clns)as 84.0 20.6 54.6 22.8 37. 511.8 28.0 9.9 (m5Cln)dAL96 A-138987.1 154usascs(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns)cs 75.5 4.8 57.4 10.6 29.5 4.219.6 2.5 (m5Clns)adAL96 A-138988.1 155gsusascs(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns) 63.1 6.9 58.2 15.3 24.0 5.121.6 5.6 cs(m5Cln)dAL96 A-138989.1 156asgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs(Tlns) 43.8 3.5 38.1 6.3 18.3 1.518.2 4.3 (Tlns)cdAL96 A-138990.1 157csasgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs(Tlns) 29.7 12.3 26.2 1.8 18.13.4 15.5 3.1 (Tln)dAL96 A-138991.1 158us(m5Clns)asgs(Tlns)ascs(m5Clns)us(Tlns)asgsasgs 34.0 7.2 31.1 7.8 18.23.4 18.2 1.7 (Tln)dAL96 A-138992.1 159(Tlns)us(m5Clns)asgs(Tlns)ascs(m5Clns)us(Tlns)as 37.9 7.7 31.1 6.1 20.02.3 18.4 0.7 gsasgdAL96 A-138976.3 160cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 69.2 12.9 65.4 8.8 23.03.8 21.2 3.9 (Tln)dAL96 A-138993.1 161cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 59.3 14.8 65.1 6.8 25.05.0 20.8 3.4 (Tlns)dAL96 A-138994.1 162cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 46.2 10.7 59.1 14.921.9 2.8 22.1 2.4 (Tlns)aL96 A-138995.1 163cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 52.7 5.8 53.4 6.9 20.94.9 20.1 2.6 (Tlns)asL96 A-138996.1 164cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 65.6 8.3 50.6 1.9 34.913.2 28.0 5.5 (Tln)dTL96 A-138997.1 165cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 79.9 6.4 56.8 6.5 41.212.8 29.3 10.0 (Tln)dGL96 A-138998.1 166cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 50.7 5.9 42.9 8.0 32.53.2 23.6 1.8 (Tln)dCL96 A-138976.4 167cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 83.3 24.1 69.5 6.1 28.03.5 26.1 6.0 (Tln)dAL96 A-138999.1 168(m5Clns)(m5Clns)us(Tlns)asgsasgs(Tlns)(Tlns)cscs 113.8 15.2 78.2 12.631.6 8.1 18.7 4.1 ascsudAL96 A-139000.1 169cscs(Tlns)(Tlns)asgsasgs(Tlns)(Tlns)cs(m5Clns) 98.7 14.8 73.6 2.5 20.97.2 19.4 1.5 ascsudAL96 A-139001.1 170(m5Clns)csus(Tlns)asgsasgs(Tlns)uscs(m5Clns) 82.2 25.7 77.3 5.9 32.0 5.121.3 8.9 ascs(Tln)dAL96 A-139002.1 171cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 29.6 11.0 44.1 14.716.3 1.7 17.6 4.8 (Tln) A-139003.1 172cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln) 20.8 4.5 29.7 5.6 11.8 3.917.3 2.7 A-139004.1 173 cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns)29.1 8.8 51.6 7.2 18.2 4.8 20.8 4.2 (Tln) A-139005.1 174cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 27.1 3.9 36.0 13.1 14.54.0 20.9 3.3 (Tln)dAL10 A-139006.1 175cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln)dAL10 44.0 14.0 40.9 4.4 18.73.9 22.0 4.7 A-139007.1 176cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns) 28.1 12.2 29.1 3.6 24.26.6 32.8 3.2 (Tln)dAL10 A-139008.1 177Q173Q173cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns) 81.3 7.7 65.0 5.5 30.47.5 21.3 3.4 as(m5Clns)(Tln) A-139009.1 178cscsuuagag(Tln)(Tln)c(m5Cln)a(m5Clns)(Tln)dAL96 96.7 12.8 65.9 8.6 55.98.6 26.8 6.1 A-139010.1 179cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Clns) 105.5 15.8 73.1 9.4 41.514.9 28.2 8.6 (Tln)dAL96 A-139011.1 180cscsususasgsasgs(Tln)(Tln)cs(m5Cln)as(m5Cln) 103.1 20.5 76.2 13.9 30.76.3 22.4 5.4 (Tln)dAL96 A-139012.1 181cscsususasgsasgsususY5scsascsudAL96 67.3 9.4 46.7 5.6 21.4 4.3 19.4 1.7A-139013.1 182 cscsususasgsasgsususcsY5sascsusdAL96 67.6 12.9 46.5 5.326.8 5.7 18.0 3.0 A-139014.1 183 cscsususasgsasgsususcscsasY5sudAL9637.0 13.2 34.9 12.8 23.6 6.2 18.6 2.2 A-139015.1 184csY5sususasgsasgsususcscsascsudAL96 68.4 13.9 61.3 9.9 20.8 5.0 19.9 2.8A-139016.1 185 (m5Clns)csususasgsasgsususcscsascsudAL96 44.2 8.7 51.28.7 18.6 3.2 20.7 1.7 A-139017.1 186cs(m5Clns)ususasgsasgsususcscsascsudAL96 64.5 4.1 55.7 7.6 24.0 3.6 23.34.5 A-139018.1 187 cscs(Tlns)usasgsasgsususcscsascsudAL96 54.1 15.2 41.43.8 20.9 8.1 24.6 2.5 A-139019.1 188cscsus(Tlns)asgsasgsususcscsascsudAL96 59.2 13.0 52.1 4.5 26.4 6.6 29.36.7 A-139020.1 189 cscsusus(Alns)gsasgsususcscsascsudAL96 54.4 25.0 45.67.4 23.1 7.9 19.8 2.1 A-139021.1 190cscsususas(Glns)asgsususcscsascsudAL96 69.0 6.4 60.3 8.4 33.2 8.9 19.33.6 A-139022.1 191 cscsususasgs(Alns)gsususcscsascsudAL96 57.2 6.5 42.713.2 36.7 12.0 22.5 5.7 A-139023.1 192cscsususasgsas(Glns)ususcscsascsudAL96 58.9 4.9 43.2 10.4 22.6 4.5 21.96.1 A-139024.1 193 cscsususasgsasgs(Tlns)uscscsascsudAL96 70.6 12.4 48.23.3 24.1 2.9 19.1 3.6 A-139025.1 194cscsususasgsasgsus(Tlns)cscsascsudAL96 64.7 24.2 49.5 8.1 25.5 6.7 16.22.5 A-139026.1 195 cscsususasgsasgsusus(m5Clns)csascsudAL96 99.4 10.271.4 10.6 35.6 5.2 20.0 3.1 A-139027.1 196cscsususasgsasgsususcs(m5Clns)ascsudAL96 59.2 8.7 51.8 8.5 22.6 6.8 16.63.1 A-139028.1 197 cscsususasgsasgsususcscs(Alns)csudAL96 45.6 8.7 46.815.6 22.9 4.6 20.2 2.3 A-139029.1 198cscsususasgsasgsususcscsas(m5Clns)usdAL96 42.6 4.2 39.5 12.5 20.0 4.720.5 4.5 A-139030.1 199 cscsususasgsasgsususcscsascs(Tln)dAL96 36.9 5.130.7 3.4 21.2 5.0 25.2 3.7 A-139031.1 200cscsususasgsasgs(Tlns)(m5Clns)gs(m5Clns) 28.3 1.6 19.2 3.4 19.1 1.9 31.37.3 us(m5Clns)(m5Cln)dAL96 A-139032.1 201cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)us(m5Clns) 46.1 10.7 46.2 13.829.6 6.6 20.0 2.3 (m5Cln)dAL96 A-139033.1 202cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)as(m5Clns) 46.9 19.5 48.5 3.8 26.96.5 23.1 6.1 (m5Cln)dAL96 A-139034.1 203cscsususasgsasgs(Tlns)(Tlns)cs(m5Clns)us(m5Clns) 30.8 11.4 40.7 12.423.6 3.5 21.6 5.5 (Tln)dAL96 A-139035.1 204gsasgsasususcscs(m5Clns)(m5Clns)us(m5Clns)cs 23.3 3.3 18.1 3.7 20.9 1.718.2 2.2 (Tlns)(m5Cln)dAL96 A-139036.1 205(m5Clns)(Tlns)usasgsasgsususcscsas(m5Clns)(Tln) 80.6 3.9 45.7 20.1 32.812.7 20.6 2.3 dAL96 A-139037.1 206(m5Clns)(Tlns)dTsdAsdGsdAsdGsdTsdTsdCsdCsd 27.5 1.9 23.3 0.7 21.3 3.518.0 1.3 As(m5Clns)(Tln)dAL96 A-139038.1 207(m5Clns)asgs(Tlns)(Alns)cscsususasgsasgsususcs 82.9 23.8 76.1 12.8 34.56.3 22.1 1.6 (m5Clns)as(m5Clns)(Tln)dAL96 A-139039.1 208(m5Clns)asgs(Tlns)(Alns)dCsdCsdTsdTsdAsdGsdA 73.9 12.3 64.8 9.1 23.9 5.720.7 5.0 sdGsdTsdTscs(m5Clns)as(m5Clns)(Tln)dAL96 AD-66568 209CfsasGfuAfcCfuUfAfGfaGfuticCfaCfuAfL96 101.9 21.1 100.1 5.7 100.2 7.3100.6 11.6 210 usAfsgUfgGfaAfalfcuaAfgGfuAfcUfgsasa

Example 2: In Vitro Reversal of Antithrombin Knockdown by Free Uptake inPrimary Mouse Hepatocytes

In vitro assay: siRNA Transfection followed by REVERSIR Free Uptake:Mouse primary hepatocytes were transfected with 1 nM siRNA by adding 4.9μL of Opti-MEM plus 0.1 L of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μL siRNA per well into a 384-wellplate and incubated at room temperature for 15 minutes. 40 μL ofWilliam's media containing ˜5×10³ cells were then added to the siRNAmixture, yielding a final siRNA concentration of 1 nM. Cells wereincubated at 37° C. After 4 h, hepatocytes were washed and REVERSIRcompounds were added by free uptake in 50 μL media for 48 h at 37° C.

Total RNA isolation using DYNABEADS mRNA Isolation Kit: RNA was isolatedfrom hepatocytes by using an automated protocol on a BioTek-EL406platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μL ofLysis/Binding Buffer and 25 μL of lysis buffer containing 3 μL ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μL Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μL Elution Buffer, re-capturedand supernatant removed.

cDNA synthesis using ABI High capacity cDNA reverse transcription kit(Applied Biosystems, Foster City, Calif., Cat #4368813): 10 μL of amaster mix containing 1 μL 10× Buffer, 0.4 μL 25×dNTPs, 1 μL 10× Randomprimers, 0.5 μL Reverse Transcriptase, 0.5 μL RNase inhibitor and 6.6 μLof water per reaction was added to RNA isolated above. Plates weresealed, mixed, and incubated on an electromagnetic shaker for 10 minutesat room temperature, followed by 2 h 37° C.

Real time PCR: 2 μL of cDNA were added to a master mix containing 0.5 μLof GAPDH TaqMan Probe, 0.5 μL RVR probe (Mm01302526_m1) and 5 μLLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well plates (Roche cat #04887301001). Real time PCR was done in aLightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay.Each REVERSIR was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. Results areshown in FIGS. 3-14.

Example 3: In Vitro Reversal of Factor IX Knockdown

Results are shown in FIGS. 15-21.

Example 4: REVERSIR Compounds are Well Tolerated In Vivo

As shown in FIG. 23, the various REVERSIR compounds tested for in vivotoxicity showed little or no change in body weight gain. Further, noliver enzyme elevation was observed across dosses, e.g., 20 and 100mg/kg. Moreover, no liver enzyme elevation was observed across timepoints, e.g., day 4 and day 8. Thus, the REVERSIR compounds of theinvention have good in vivo tolerability and safety profile.

Example 5: Reversal of Antithrombin Knockdown by REVERSIR Compounds inNon-Human Primates

A single dose exploratory pharmacology study of REVERSIR compounds wascarried out in male Cynomolgus monkeys.

Experimental Design

Animals judged to be suitable for testing were arbitrarily assigned tothe study and arranged in seven groups as shown in Table 9. One day 0(the first day of dosing), all animals received a single subcutaneousdose of antithrombin siRNA (ALN-AT3SC). The pharmacology and toxicologyof ALN-AT3SC has previously been evaluated in rodents, dog, rabbit andnon-human primate (cynomolgus monkey). Thus, single administration of7.5 mg/kg was expected to be well-tolerated with no adversephysiological or histological effects.

TABLE 9 ALN-AT3SC Dose Number Group Dose Level Concentration Volume ofNumber (mg/kg) (mg/mL) (mL/kg) Males 1 7.5 3.75 2 3 2 7.5 3.75 2 3 3 7.53.75 2 3 4 7.5 3.75 2 3 5 7.5 3.75 2 3 6 7.5 3.75 2 3 7 7.5 3.75 2 3

One day 14, animals received a single subcutaneous dose of REVERSIRcompounds (A-138959, A-140340 or A-140337) or saline as shown in Table10. The dose levels of the REVERSIR compounds replicated levelspreviously evaluated in subcutaneous pharmacokinetic/pharmacodynamicstudies in mice.

TABLE 10 Test Article Dose Dose Group Level Concentration Volume Numberof Number Test Article (mg/kg (mg/mL) (mL/kg) Males 1 0.9% Saline 0 0 23 2 A-138959 0.25 0.125 2 3 3 A-138959 2.5 1.25 2 3 4 A-140340 0.250.125 2 3 5 A-140340 2.5 1.25 2 3 6 A-140337 0.25 0.125 2 3 7 A-1403372.5 1.25 2 3

All subcutaneous doses were performed with the appropriately sizedsyringe per standard operating procedure. Following administration onday 14, animals were maintained on study for a 6-week non-dosing period.

Pharmacokinetics

Animals receiving the REVERSIR compounds were bled 2 and 8 hourspost-dose on day 14 and once on days 15, 16, 17, 18 and 21. About 1.0 mlof blood (per time point) was collected from the femoral or othersuitable vein. K₂EDTA was used as the anticoagulant. Samples were keptchilled (wet ice, as appropriate) during collection and duringprocessing. The samples were centrifuged 2400-2700 rpm at approximately4° C. for approximately 10 minutes. The maximum amount of plasmarecovered was divided into two approximately equal volume aliquots intoappropriately labeled tubes. Aliquoted sample were designated as“Aliquot 1” or “Aliquot 2”. Both sets of samples were maintained frozen(−65° C. to −85° C.) until bioanalytical evaluation.

Pharmacodynamics (Plasma AT)

All animals were bled twice during pretest (Days −5 and −2) and on days0, 7, 14, 15, 16, 17, 18, 21, 28, 35, 42, 49, and 56. About 1.0 ml ofblood (per time point) was collected from the femoral or other suitablevein. K₂EDTA was used as the anticoagulant. Samples were kept chilled(wet ice, as appropriate) during collection and during processing. Thesamples were centrifuged 2400-2700 rpm at approximately 4° C. forapproximately 10 minutes. The maximum amount of plasma recovered wasdivided into two approximately equal volume aliquots into appropriatelylabeled tubes. Aliquoted sample were designated as “Aliquot 1” or“Aliquot 2”. Both sets of samples were maintained frozen (−65° C. to−85° C.) until evaluation of plasma antithrombin via ELISA.

Immunostimulation (Plasma Cytokines)

All animals receiving the REVERSIR compounds on day 14 prior to dosingand at 4 and 24 hours post-dosing. About 0.6 ml of blood (per timepoint) was collected from the femoral or other suitable vein. K₂EDTA wasused as the anticoagulant. Samples were kept chilled (wet ice, asappropriate) during collection and during processing. The samples werecentrifuged 2400-2700 rpm at approximately 4° C. for approximately 10minutes. The maximum amount of plasma recovered was divided into twoapproximately equal volume aliquots into appropriately labeled tubes.Aliquoted sample were designated as “Aliquot 1” or “Aliquot 2”. Bothsets of samples were maintained frozen (−65° C. to −85° C.) untilevaluation of plasma cytokines/chemokines by multiplex assay.

Clinical pathology: During pretest and on days 16 and 56, varioushematology and serum chemistry parameters were evaluated from allanimals (Groups 1-7) once during the pretest period and on days 16 and56 as a clinical pathology screen for the purpose of animalselection/confirmation of health. Blood samples were collected fromfasted animals via a femoral vein (or other suitable vein). Theanticoagulant used was K₂EDTA for the hematology samples and sodiumcitrate for the coagulation samples. Samples for serum chemistry werecollected without anticoagulant.

Hematology parameters included differential leukocyte count, erythrocytecount, hemoglobin, hemoglobin distribution width hematocrit, meancorpuscular hemoglobin, mean corpuscular volume, mean corpuscularhemoglobin concentration, platelet count, red cell distribution width,reticulocyte count and total leukocyte count.

Serum chemistry parameters included alanine aminotransferase, albumin,alkaline phosphatase, albumin/globulin ratio (calculated), aspartateaminotransferase, calcium chloride, creatinine, gammaglutamyltransferase, globulin (calculated), glucose, phosphorus,potassium, sodium, sorbitol dehydrogenase, total bilirubin, totalcholesterol, total protein, triglycerides, urea nitrogen, andappearance.

As shown in FIGS. 24A and 24B, the tested exemplary REVERSIR compoundsreversed the activity of the antithrombin siRNA in non-human primates.In FIG. 24A, REVERSIR compounds were administered at a dose of 2.5 mg/kg(0.75 molar eq. of the siRNA, ALN-AT3). In FIG. 24B, REVERSIR compoundswere administered at a dose of 0.25 mg/kg (0.075 molar eq. of the siRNA,ALN-AT3). Surprisingly, REVERSIR A-140340 (a 9-mer with lowphosphorothioate content, 5 PS) showed complete reversal of ALN-AT3activity within 4 days of dosing and was active at 30-fold lower dosethan the conjugate (13 molar eq.).

Abbreviations used in describing the sequences, e.g., sequencesdescribed in Tables 2-8 are collected and described in Table 11 forconvenience.

TABLE 11 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Af2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cf2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioateCs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gbbeta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioateGf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dT 2′-deoxythymidine dTs2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine sphosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos)2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo)2′-O-methoxyethylguanosine-3′-phosphate (Geos)2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo)2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos)2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo)2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos)2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate (A3m)3′-O-methyladenosine-2′-phosphate (A3mx)3′-O-methyl-xylofuranosyladenosine-2′-phosphate (G3m)3′-O-methylguanosine-2′-phosphate (G3mx)3′-O-methyl-xylofuranosylguanosine-2′-phosphate (C3m)3′-O-methylcytidine-2′-phosphate (C3mx)3′-O-methyl-xylofuranosylcytidine-2′-phosphate (U3m)3′-O-methyluridine-2′-phosphate (U3mx)3′-O-methylxylouridine-2′-phosphate (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (pshe) Hydroxyethylphosphorothioate(Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Tgn) Thymidine-glycol nucleicacid (GNA) S-Isomer (Cgn) Cytidine-glycol nucleic acid (GNA) (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (Ggn)2′-O-hexadecyl-cytidine-3′-phosphate (Agn) Adenosine-glycol nucleic acid(GNA) P 5′-phosphate (m5Cam)2′-O-(N-methylacetamide)-5-methylcytidine-3′-phosphate (m5Cams)2′-O-(N-methylacetamide)-5-methylcytidine-3′-phosphorothioate (Tam)2′-O-(N-methylacetamide)thymidine-3′-phosphate (Tams)2′-O-(N-methylacetamide)thymidine-3′-phosphorothioate (Aam)2′-O-(N-methylacetamide)adenosine-3′-phosphate (Aams)2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate (Gam)2′-O-(N-methylacetamide)guanosine-3′-phosphate (Gams)2′-O-(N-methylacetamide)guanosine-3′-phosphorothioate Y442-hydroxymethyl-tetrahydrofurane-5-phosphate Q173N-((GalNAc)-amidopentanoyl)-prolinol-4-phosphate (Hyp-C5-(GalNAc))

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

What is claimed is: 1.-3. (canceled)
 4. A method of inhibiting theactivity of a siRNA in a cell comprising contacting the cell with aREVERSIR compound comprising a modified oligonucleotide consisting of 6to 25 linked nucleotides and having a nucleobase sequence substantiallycomplementary to antisense strand of the siRNA. 5.-13. (canceled)
 14. Amethod of treating a subject comprising: administering to the subject asiRNA; monitoring the subject for siRNA activity; if the siRNA activitybecomes higher than desired, administering a REVERSIR compound to thesubject. 15.-27. (canceled)
 28. A REVERSIR compound comprising amodified oligonucleotide consisting of 6 to 20 linked nucleotides andhaving a nucleobase sequence substantially complementary to antisensestrand of a siRNA.
 29. The REVERSIR compound of claim 28, wherein themodified oligonucleotide consists of 8-15 linked nucleotides.
 30. TheREVERSIR compound of claim 28, wherein the modified oligonucleotide is asingle-stranded oligonucleotide having at least 90% complementary to theantisense strand.
 31. The REVERSIR compound of claim 28, wherein themodified oligonucleotide is substantially complementary to nucleotides2-16 of the antisense stand.
 32. The REVERSIR compound of claim 28,wherein the modified oligonucleotide is fully complementary to theantisense strand.
 33. The REVERSIR compound of claim 28, wherein themodified oligonucleotide comprises at least one modified internucleotidelinkage.
 34. The REVERSIR compound of claim 28, wherein the modifiedoligonucleotide comprises at least one modified internucleotide linkageand at least one unmodified internucleotide linkage.
 35. The REVERSIRcompound of claim 34, wherein the modified oligonucleotide comprises anunmodified internucleotide linkage between the 3′-terminus nucleotideand the penultimate nucleoside.
 36. The REVERSIR compound of claim 28,wherein the modified oligonucleotide comprises at least one modifiednucleobase.
 37. The REVERSIR compound of claim 28, wherein the modifiedoligonucleotide comprises at least one modified sugar.
 38. The REVERSIRcompound of claim 37, wherein said at least one modified sugar is abicyclic sugar.
 39. The REVERSIR compound of claim 28, wherein themodified oligonucleotide comprises at least one nucleotide wherein 2′position of furnaosyl is connected to the 4′ position by a linkerselected independently from —[C(R1)(R2)]_(n)-, —[C(R1)(R2)]_(n)-O—,—[C(R1)(R2)]_(n)—N(R1)-, —[C(R1)(R2)]_(n)—N(R1)-O—,[C(R1R2)]_(n)-O—N(R1)-, —C(R1)=C(R2)-O—, —C(R1)=N—, —C(R1)=N—O—,C(═NR1)-, C(═NR1)-O—, C(═O)—, C(═O)O—, C(═S)—, C(═S)O—, C(═S)S—, O,Si(R1)2-, S(═O)_(x)— and N(R1)-, wherein: x is 0, 1, or 2; n is 1, 2, 3,or 4; each R1 and R2 is, independently, H, a protecting group, hydroxyl,C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substitutedC2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,substituted C5-C20 aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical,substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3,COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), orsulfoxyl (S(═O)-J1); and each J1 and J2 is, independently, H, C1-C12alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C1-C12 aminoalkyl,substituted C1-C12 aminoalkyl or a protecting group.
 40. The REVERSIRcompound of claim 28, wherein the modified oligonucleotide is conjugatedwith a ligand.
 41. The REVERSIR compound of claim 28, wherein themodified oligonucleotide is conjugated with a ligand of structure:


42. The REVERSIR compound of claim 28, wherein the modifiedoligonucleotide is conjugated with a ligand and the ligand is conjugatedto 3′-terminus of the modified oligonucleotide.
 43. The REVERSIRcompound of claim 28, wherein the modified oligonucleotide is conjugatedwith a ligand and the ligand is conjugated to a nucleoside with a deoxysugar in the REVERSIR compound.
 44. The REVERSIR compound of claim 43,wherein said deoxy sugar is a 2′-deoxy ribose.
 45. The REVERSIR compoundof claim 28, wherein the siRNA is targeted to an mRNA, a pre-mRNA, amicro-RNA a pre-micro-RNA. 46.-48. (canceled)